Variety corn line HAF4561

ABSTRACT

The present invention provides an inbred corn line designated HAF4561, methods for producing a corn plant by crossing plants of the inbred line HAF4561 with plants of another corn plant. The invention further encompasses all parts of inbred corn line HAF4561, including culturable cells. Additionally provided herein are methods for introducing transgenes into inbred corn line HAF4561, and plants produced according to these methods.

REFERENCE TO RELATED APPLICATION

This application claims the priority benefit under Title 35, UnitedStates Code 119(e) of U.S. Provisional Patent Application No. 61/769,951filed Feb. 27, 2013.

FIELD OF THE INVENTION

This invention is in the field of corn breeding. Specifically, thepresent invention provides a maize plant and its seed designatedHAF4561, as well as derivatives and hybrids thereof.

BACKGROUND OF THE INVENTION

Maize (or corn; Zea mays L.) plant breeding is a process to developimproved maize germplasm in an inbred or hybrid plant. Maize plants canbe self-pollinating or cross pollinating. Self pollination for severalgenerations produces homozygosity at almost all gene loci, forming auniform population of true breeding progeny, known as inbreds. Hybridsare developed by crossing two homozygous inbreds to produce heterozygousgene loci in hybrid plants and seeds. In this process, the inbred isemasculated and the pollen from the other inbred pollinates theemasculated inbred. Emasculation of the inbred can be done by chemicaltreatment of the plant, detasseling the seed parent, or the parentinbred can comprise a male sterility trait or transgene impartingsterility, eliminating the need for detasseling. This emasculatedinbred, often referred to as the female, produces the hybrid seed, F1.The hybrid seed that is produced is heterozygous. However, the grainproduced by a plant grown from F1 hybrid seed is referred to as F2grain. F2 grain which is a plant part produced on the F1 plant willcomprise segregating maize germplasm, even though the hybrid plant isheterozygous.

Such heterozygosity in hybrids results in robust and vigorous plants.Inbred plants on the other hand are mostly homozygous, rendering themless vigorous. Inbred seed can be difficult to produce due to suchdecreased vigor. However, when two inbred lines are crossed, theresulting hybrid plant shows greatly increased vigor and seed yieldcompared to open pollinated, segregating maize plants. An importantconsequence of the homozygosity and homogeneity of inbred maize lines isthat all hybrid seed and plants produced from any cross of two suchlines will be the same. Thus the use of inbreds allows for theproduction of hybrid seed that can be readily reproduced.

There are numerous stages in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possess the traits to meetthe program goals. The aim is to combine in a single variety an improvedcombination of desirable traits from the parental germplasm. Theseimportant traits may include, for example, higher yield, resistance todiseases, fungus, bacteria and insects, better stems and roots,tolerance to drought and heat, improved nutritional quality, and betteragronomic characteristics.

Choice of breeding methods depends on the mode of plant reproduction,the heritability of the trait(s) being improved, and the type ofcultivar used commercially (e.g., F1 hybrid cultivar, pure linecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location may be effective,whereas for traits with low heritability, selection can be based on meanvalues obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences the choice of breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars andintroducing transgenic events into maize germplasm. Thus, backcrossbreeding is useful for transferring genes for a simply inherited, highlyheritable trait into a desirable homozygous cultivar or inbred linewhich is the recurrent parent. The source of the trait to be transferredis called the donor parent. After the initial cross, individualspossessing the phenotype of the donor parent are selected and repeatedlycrossed (backcrossed) to the recurrent parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent.

Each breeding program generally includes a periodic, objectiveevaluation of the efficiency of the breeding procedure. Evaluationcriteria vary depending on the goals and objectives, but should includegain from selection per year based on comparisons to an appropriatestandard, overall value of the advanced breeding lines, and number ofsuccessful cultivars produced per unit of input (e.g., per year, perdollar expended, etc.).

The ultimate objective of commercial corn breeding programs is toproduce high yield, agronomically sound plants that perform well inparticular regions of the U.S. Corn Belt, such as a plant of thisinvention.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a seed of the maizevariety HAF4561, representative seed of said variety having beendeposited.

In a further aspect, the present invention provides a plant of maizevariety HAF4561, representative seed of said HAF4561 variety having beendeposited. Further provided is a plant part of the plant of thisinvention, which includes but is not limited to a pollen grain, a silk,a protoplast, a cell, a tassel, an anther, or an ovule. Also provided isa maize seed produced on the plant of maize variety HAF4561.

Another aspect of the invention provides a maize plant having all thephysiological and morphological characteristics of maize variety HAF4561and further comprising an additional trait, wherein the additional traitis selected from the group consisting of increased water stressresistance, waxy starch, male sterility or restoration of malefertility, modified carbohydrate metabolism, modified protein metabolismand modified fatty acid metabolism, altered starch, thermotolerantamylase, herbicide resistance, insect resistance, nematode resistance,bacterial disease resistance, fungal disease resistance, and viraldisease resistance. In some embodiments, the additional trait isconferred by introducing a transgene.

Additional aspects of this invention include a process for producing anF1 hybrid maize seed, said process comprising crossing a plant of maizeinbred plant HAF4561 with a different maize plant and harvesting theresultant F1 hybrid maize seed. A maize plant or plant part produced bygrowing the F1 hybrid maize seed is also provided herein. The presentinvention also provides a maize seed produced by crossing the plant ofthis invention with a different maize plant.

The present invention further provides an F1 hybrid maize seedcomprising an inbred maize plant cell of inbred maize plant HAF4561.Further provided is a process of crossing the maize inbred plant HAF4561with another plant. Additionally provided herein is the seed produced bycrossing the maize inbred plant HAF4561 with another plant.Additionally, a plant produced by germinating the seed produced bycrossing the maize inbred plant HAF4561 with another plant is provided

A method is also provided for producing maize seed comprising growingthe plant of this invention until seed is produced and harvesting theseed, wherein the harvested seed is inbred or hybrid or haploid seed.

The present invention also provides a method of producing seed,comprising crossing the plant of the invention with itself or a secondmaize plant. Seed produced by this method are also provided herein.Additional aspects of this invention include hybrid seed produced bycrossing the invention with a second distinct corn plant and the plantand plant parts on this hybrid plant grown from the hybrid seed.

Additional aspects of this invention include a process of introducing anadditional trait into maize inbred plant HAF4561, comprising: (a)crossing HAF4561 plants grown from HAF4561 seed with plants of anothermaize plant that comprise an additional trait to produce hybrid progenyplants, (b) selecting hybrid progeny plants that have the additionaltrait to produce selected hybrid progeny plants; (c) crossing theselected progeny plants with the HAF4561 plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have theadditional trait to produce selected backcross progeny plants; and (e)repeating step(s) (c) and (d) one or more times to produce backcrossprogeny plants of subsequent generations that comprise the additionaltrait and all of the physiological and morphological characteristics ofmaize inbred plant HAF4561 when grown in the same environmentalconditions. In some embodiments of this invention, the desired trait canbe, but is not limited to, waxy starch, male sterility, increasedtolerance to water stress, herbicide resistance, nematode resistance,modified amylase, altered starch, thermotolerant amylase, insectresistance, modified carbohydrate metabolism, protein metabolism, fattyacid metabolism, bacterial resistance, disease resistance, fungaldisease resistance, viral disease resistance, or any combinationthereof. A plant produced by this process is also provided herein. Or aconversion of this maize variety, wherein representative seed of saidmaize variety comprising at least one new trait wherein said conversionshad the morphological and physiological traits of maize and said traitconfers a characteristic selected from the group consisting of alteredamylase, abiotic stress and biotic stress tolerance, herbicide, insect,fungal, bacterial and disease resistance.

Furthermore, the present invention provides a maize plant having all thephysiological and morphological characteristics of inbred plant HAF4561,wherein a sample of the seed of inbred plant HAF4561 was deposited. Themaize plant of this invention can comprise a genome which furthercomprises at least one transgene and/or the maize plant can exhibit atrait conferred by a transgene. In some embodiments of this invention,the transgene can confer a trait of herbicide resistance or tolerance;insect resistance or tolerance; resistance or tolerance to bacterial,fungal, nematode or viral disease; waxy starch; altered starch, malesterility or restoration of male fertility, modified carbohydratemetabolism, modified fatty acid metabolism, or any combination thereof.

Additionally provided herein is a method of producing a maize plantderived from the inbred plant HAF4561, comprising the steps of: (a)growing a progeny plant wherein the inbred plant is one parent of theprogeny; (b) crossing the progeny plant with itself or a different plantto produce a seed of a progeny plant of a subsequent generation; (c)growing a progeny plant of a subsequent generation from said seed andcrossing the progeny plant of a subsequent generation with itself or adifferent plant; and (d) repeating steps (b) and (c) for an additional0-5 generations to produce a maize plant derived from the inbred plantHAF4561.

Another aspect of this invention includes a method for developing amaize plant in a maize plant breeding program, comprising applying plantbreeding techniques comprising recurrent selection, backcrossing,pedigree breeding, marker enhanced selection, haploid/dihaploidproduction, or transformation to the maize plant of this invention, orits parts, wherein application of said techniques results in developmentof a maize plant.

Furthermore, the present invention provides a method of producing acommodity plant product comprising growing the plant from the seed ofthis invention or a part thereof and producing said commodity plantproduct, wherein said commodity plant product can be, but is not limitedto a protein concentrate, a protein isolate, starch, meal, flour, oiltherefrom, or any combination thereof.

A method is also provided of producing a treated seed of this invention,comprising obtaining the seed of HAF4561 and treating said seed.According to one aspect, the present invention provides a method ofproducing a genetic marker profile comprising extracting nucleic acidsfrom the seed produced by maize variety HAF4561 or the plant grown fromsaid seed and genotyping said nucleic acids, thereby producing a geneticmarker profile.

According to another aspect, the present invention provides a method ofplant breeding comprising: isolating nucleic acids from a seed producedby maize variety HAF4561 or a plant grown from the seed, identifying oneor more polymorphisms from the isolated nucleic acids, and selecting aplant having one or more poly morphisms wherein the plant is used in aplant breeding method.

In a still further aspect, the present invention provides a method ofproducing an inbred maize plant derived from the inbred maize varietyHAF4561 the method comprising: crossing a progeny plant wherein theinbred plant HAF4561 is one parent of the progeny, wherein said plantmay have one or more traits, with an inducer maize plant to producehaploid seed; and doubling the haploid seed to produce an inbred maizeplant.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. The terminology used in thedescription of the invention herein is for the purpose of describingparticular embodiments only and is not intended to be limiting of theinvention. All publications, patent applications, patents, and otherreferences mentioned herein are incorporated by reference in theirentirety.

In the description and examples that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecifications and claims, including the scope to be given such terms,the following definitions are provided.

DEFINITIONS OF PLANT CHARACTERISTICS

Early Season Trait Codes

Emergence Rating (EMRGR): Recorded when 50% of the plots in the trialare at V1 (1 leaf collar) growth stage. Various responses include, butare not limited to, (1) All plants have emerged and are uniform in size;(2) All plants have emerged but are not completely uniform; (3) Mostplants have emerged with some just beginning to break the soil surface,noticeable lack of uniformity; (4) Less than 50% of the plants haveemerged, and lack of uniformity is very noticeable; or (5) A few plantshave emerged but most remain under the soil surface.

Seedling Growth (SVGRR or Vigor): Recorded between V3 and V5 (3-5 leafstage) giving greatest weight to seedling plant size and secondaryweight to uniform growth. Various responses include, but are not limitedto, (1) Large plant size and uniform growth; (2) Acceptable plant sizeand uniform growth; (3) Acceptable plant size and might be a littlenon-uniform; (4) Weak looking plants and non-uniform growth; or (5)Small plants with poor uniformity.

Purpling (PRPLR): Emergence and/or early growth rating. Purpling is morepronounced on the under sides of leaf blades especially on midribs.Various responses include, but are not limited to, (1) No plants showingpurple color; (2) 30% plants showing purple color; (3) 50% plantsshowing purple color; (4) 70% plants showing purple color; or (5) 90+%plants showing purple color.

Herbicide Injury (HRBDR): List the herbicide type that is being rated.Then rate each hybrid/variety injury as indicated below. (1) No apparentreduction in biomass or other injury symptoms; (2) Moderate reduction inbiomass with some signs of sensitivity; (3) Severe reduction in biomasswith some mortality.

Mid-Season Trait Codes

Heat Units to 50% Silk (HU5SN): Recorded the day when 50% of all plantswithin a plot show 2 cm or more silk protruding from the ear. Converteddays to accumulated heat units from planting.

Heat units to 50% Pollen Shed (HUPSN): Recorded the day when 50% of allplants within a plot are shedding pollen. Converted days to accumulatedheat units from planting.

Plant Height (PLHTN): After pollination, recorded average plant heightof each plot. Measured from ground to tassel tip in cm.

Plant Ear Height (ERHTN) in cm: After pollination, record average earheight of each plot. Measure from ground to base of ear node (shank).

Root Lodging Early % (ERTLP): Early root lodging occurs up to about twoweeks after flowering and usually involves goosenecking. The number ofroot lodged plants are counted and converted to a percentage.

Shed Duration (Shed Duration): Sum of daily heat units for days whenplants in the plot are actively shedding pollen.

Foliar Disease (LFDSR): Foliar disease ratings taken one month beforeharvest and through harvest. The predominant disease should be listed inthe trial information and individual hybrid ratings should be given.Various responses include, but are not limited to, (9) No lesions to twolesions per leaf; (8) A few scattered lesions on the leaf. About five toten percent of the leaf surface is affected; (7) A moderate number oflesions are on the leaf. About 15 to 20 percent of the leaf surface isaffected; (6) abundant lesions are on the leaf. About 30 to 40 percentof the leaf surface is affected; or (5) Highly abundant lesions (>50percent) on the leaf. Lesions are highly coalesced. Plants may beprematurely killed. Alternatively, the response to diseases can also berated as: R=Resistant=9 to 8 rating; MR=Moderately Resistant=7 to 6rating; MS=Moderately Susceptible=5 to 4 rating; S=Susceptible=3 to 1rating

Preharvest Trait Codes

Heat units to Black Layer (HUBLN): The day when 50% of all plants withina plot reach the black layer stage is recorded. Convert days toaccumulated heat units from planting.

Harvest Population (HAVPN): The number of plants in yield rows,excluding tillers, in each plot is counted.

Barren Plants (BRRNP): The number of plants in yield rows having no earsand/or abnormal ears with less than 50 kernels is counted.

Dropped Ears (DROPP): The numbers of ears lying on the ground in yieldrows are counted.

Stalk Lodging % (STKLP): Stalk lodging will be reported as number ofplants broken below the ear without pushing, excluding green snappedplants. The number of broken plants in yield rows is counted andconverted to percent.

Root Lodging Late % (LRTLP): Late root lodging can usually start tooccur about two weeks after flowering and involves lodging at the baseof the plant. Plants leaning at a 30-degree angle or more from thevertical are considered lodged. The number of root lodged plants inyield rows is counted and converted to percent.

Push Test for Stalk and Root Quality on Erect Plants % (PSTSP or PCTPush or % Pushtest): The push test is applied to trials withapproximately five percent or less average stalk lodging. Plants arepushed that are not root lodged or broken prior to the push test.Standing next to the plant, the hand is placed at the top ear and pushedto arm's length. Push one of the border rows (four-row small plot) intoan adjacent plot border row. The number of plants leaning at a 30-degreeangle or more from the vertical, including plants with broken stalksprior to pushing is counted. Plants that have strong rinds that snaprather than bend over easily are not counted. The goal of the push testis to identify stalk rot and stalk lodging potential, NOT ECB injury.Data may be collected for the push test in the following manner:

PUSXN: Push ten plants and enter the number of plants that do not remainupright.

Intactness (INTLR): Responses can include, but are not limited to, (1)Healthy appearance, tops unbroken; (2) 25% of tops broken; or (3)Majority of tops broken

Plant Appearance (PLTAR): This is a visual rating based on general plantappearance, taking into account all factors of intactness, pest anddisease pressure. Various responses include, but are not limited to, (1)Complete plant with healthy appearance; (2) Plants look okay; or (3)Plants are not acceptable.

Green Snap (GRSNP or PCTGS or % GreenSnap): Count the number of plantsin yield rows that snap below the ear due to brittleness associated withhigh winds.

Stay-green (STGRP): This is an assessment of the ability of a grainhybrid to retain green color as maturity approaches (taken near the timeof black-layer formation) and should not be a reflection of hybridmaturity or leaf disease. Record as a percentage of green tissue. Thismay be listed as a Stay Green Rating instead of a percentage.

Stay Green Rating (STGRR): This is an assessment of the ability of agrain hybrid to retain green color as maturity is approached (taken nearthe time of black layer formation or if major differences are notedlater). This rating should not be a reflection of the hybrid maturity orleaf disease. Ratings are 1-9. (1=best, 9=worst) 1=solid Green Plant9=no green tissue

Ear/Kernel Rots (KRDSR): If ear or kernel rot is present, husk tenconsecutive ears in each plot and count the number that have evidence ofear or kernel rot, multiply by 10, and round up to the nearest rating asdescribed below. Identify and record the disease primarily responsiblefor the rot. The rot response can include but is not limited to (1) Norot, 0% of the ears infected; (2) Up to 10% of the ears infected; (3) 11to 20% of the ears infected; (4) 21 to 35% of the ears infected; or (5)36% or more of the ears infected.

Grain Quality (GRQUR): Observations taken on husked ears after blacklayer stage. The kernel cap integrity and relative amount of soft starchendosperm along the sides of kernels are rated. Grain quality ratingscan include but are not limited to (1) Smooth kernel caps and or 10% orless soft starch; (2) Slight kernel wrinkles and or 30% soft starch; (3)Moderate kernel wrinkles and or 70% soft starch; or (4) Severe kernelwrinkled and or 90% or more soft starch.

Preharvest Hybrid Trait Codes

Ear Shape (DESHR): Description of ear shape can include, but is notlimited to, (1) Blocky; (2) Semi-blocky; or (3) Slender.

Ear Type (EARFR): Description of ear type can include, but is notlimited to, (1) Flex; (2) Semi-flex; or (3) Fixed.

Husk Cover (HSKCR):): Description of husk cover can include, but is notlimited to, (1) Long; (2) Medium; or (3) Short.

Kernel Depth (KRLNR): Description of kernel depth can include, but isnot limited to, (1) Deep; (2) Medium; or (3) Short (shallow).

Shank Length (SHLNR): Description of shank length can include, but isnot limited to, (1) Short; (2) Medium; or (3) Long.

Kernel Row Number (KRRWN): The average number of kernel rows on 3 ears.

Cob diameter (COBDR): Cob diameter is to be taken with template.Description of cob diameter can include, but is not limited to, (1)Small; (2) Medium; or (3) Large.

Harvest Trait Codes

Number of Rows Harvested (NRHAN)

Plot Width (RWIDN)

Plot Length (RLENN)

Yield Lb/Plot (YGSMN): Bushels per acre adjusted to 15.5% moisture.

Test Weight (TSTWN or TWT): Test weight at harvest in pounds per bushel.

Moisture % (MST_P): Percent moisture of grain at harvest.

Adjusted Yield in Bu/A (YBUAN) listing of bushels per acre of harvestedseed at standard moisture

Kernel Type (KRTPN): Description of kernal type can include, but is notlimited to, (1) Dent; (2) Flint; (3) Sweet; (4) Flour; (5) Pop; (6)Ornamental; (7) Pipecorn; or (8) Other.

Endosperm Type (KRTEN): Description of endosperm type can include, butis not limited to, (1) Normal; (2) Amylose (high); (3) Waxy (4) Sweet;(5) Extra sweet; (6) High protein; (7) High lysine; (8) Super sweet; (9)High oil; or (10) Other.

Sterile Type (MSCT): Description of sterile type can include, but is notlimited to, (1) No; If yes, cytoplasm type can include but is notlimited to, (2) C-type or (3) S-type if other (4) for example, transgene

Anthocyanin of Brace Roots (PBRCC): Refers to the presence of color on60% of the brace roots during pollen shed. The description of theanthocyanin of brace roots can include, but is not limited to, (1)Absent; (2) Faint; (3) Moderate; (4) Dark; (5) Brace Roots not present;(6) Green; (7) Red; or (8) Purple.

Anther Color (ANTCC): At 50 percent pollen shed observe the color ofnewly extruded anthers, pollen not yet shed. The description of theanther color can include, but is not limited to, (1) Yellow; (2) Red;(3) Pink; or (4) Purple

Glume Color (GLMCC): Color of glumes prior to pollen shed. Thedescription of the glume color can include, but is not limited to, (1)Red or (2) Green.

Silk Color (SLKCC; SLKCN): Taken at a late flowering stage when allplants have fully extruded silk. Silks at least 2″ long but still fresh.The description of the silk color can include, but is not limited to,(1) Yellow; (2) Pink; or (3) Red (e.g., Munsell value).

Kernel Color (KERCC): The main color of the kernel from at least threeears per ear family. The description of the kernel color can include,but is not limited to, (1) Yellow; or (2) White.

Cob Color (COBCC; COBCC): The main color of the cob after shelling fromat least three ears per ear family. The description of the cob color caninclude, but is not limited to, (1) Red; (2) Pink; or (3) White (e.g.,Munsell value).

Additional Definitions Relating to Plant Culture and PlantCharacteristics

Final number of plants per plot EMRGN

Region Developed (REGNN): Various response can include, but are notlimited to, (1) Northwest; (2) Northcentral; (3) Northeast; (4)Southeast; (5) Southcentral; (6) Southwest; or (7) Other.

Cross type (CRTYN); The cross types include, but are not limited to, (1)sc 2; (2) dc; (3) 3w; (4) msc; (5) m3w; (6) inbred; (7) rel. line; or(8) Other.

Days to Emergence (EMERN).

Percent Root lodging (before anthesis) (ERTLP).

Percent Brittle snapping (before anthesis) (GRSNP).

Tassel branch angle (degree) of 2nd primary lateral branch (at anthesis)(TBANN).

Days to 50% silk in adapted zone (DSAZN).

Heat units to 90% pollen shed (from emergence) (HU9PN).

Days from 10% to 90% pollen shed (DA19N).

Heat units from 10% to 90% pollen shed (HU19N).

Heat units to 10% pollen shed: (from emergence) (HU1PN)

Leaf sheath pubescence of second leaf above the ear (at anthesis) 1-9(1=none) (LSPUR).

Angle (degree) between stalk and 2nd leaf above the ear (at anthesis)(ANGBN).

Color of second leaf above the ear (at anthesis) (CR2LN) (Munsellvalue).

Glume color bars perpendicular to their veins (glume bands) (GLCBN): canbe described as (1) absent or (2) present.

Anther color (Munsell value) (ANTCN).

Pollen Shed (PLQUR): Can be described numerically, for example, 1-9(0=male sterile).

Number of leaves above the top ear node (LAERN).

Number of lateral tassel branches that originate from the central spike(LTBRN).

Number of ears per stalk (EARPN).

Husk color (Munsell value) 25 days after 50% silk (fresh) (HSKCN).

Husk color (Munsell value) 65 days after 50% silk: (dry) (HSKDN).

Leaf marginal waves: Can be described numerically, for example, 1-9(1=none) (MLWVR).

Leaf longitudinal creases (LFLCR): Can be described numerically, forexample, 1-9 (1=none).

Length (cm) of ear leaf at the top ear node (ERLLN).

Width (cm) of ear leaf at the top ear node at the widest point (ERLWN).

Plant height (cm) to tassel tip (PLHTN).

Plant height (cm) to the top ear node (ERHCN).

Length (cm) of the internode between the ear node and the node above(LTEIN).

Length (cm) of the tassel from top leaf collar to tassel tip (LTASN).

Days from 50% silk to 25% grain moisture in adapted zone (DSGMN).

Shank length (cm) (SHLNN).

Ear length (cm) (ERLNN).

Diameter (mm) of the ear at the midpoint (ERDIN).

Weight (gm) of a husked ear (EWGTN).

Kernel rows (KRRWR): Can be described as, for example, (1) Indistinct or(2) Distinct.

Kernel row alignment (KRNAR): Can be described as, for example, (1)Straight; (2) Slightly Curved; or (3) Curved.

Ear taper (ETAPR): Can be described as, for example, (1) Slight; (2)Average; or (3) Extreme.

Number of kernel rows (KRRWN).

Husk tightness 65 days after 50% silk (HSKTR): Can be describednumerically, for example, 1-9 (1=loose).

Diameter (mm) of the cob at the midpoint (COBDN).

Yield (YKGHN) (kg/ha) Kg per Hectare.

Hard endosperm color (KRCLN) (Munsell value)

Aleurone color (ALECN) (Munsell value)

Aleurone color pattern (ALCPR): Can be described, for example, as (1)homozygous or (2) segregating.

Kernel length (mm) (KRLNN).

Kernel width (mm) (KRWDN).

Kernel thickness (mm) (KRDPN).

One hundred kernel weight (gm) (K1KHN)

Husk extension (HSKCR): Can be described as, for example, (1) Short (earexposed); (2) Medium (8 cm); (3) Long (8-10 cm); or (4) Very long (>10cm).

Percent round kernels on 13/64 slotted screen (KRPRN).

Position of ear 65 days after 50% silk (HEPSR): Can be described as, forexample, (1) Upright; (2) Horizontal; or (3) Pendent.

Percent dropped ears 65 days after anthesis (DPOPP).

Percent root lodging 65 days after anthesis (LRTRP).

Heat units to 25% grain moisture (from emergence) (HU25N).

Heat units from 50% silk to 25% grain moisture in adapted zone (HUSGN).

OTHER DEFINITIONS

A, AN, THE—As used herein, “a,” “an” or “the” can mean one or more thanone. For example, a cell can mean a single cell or a multiplicity ofcells.

AND/OR—As used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(or).

ABOUT—The term “about,” as used herein when referring to a measurablevalue such as an amount of a compound or agent, dose, time, temperature,and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%,±0.5%, or even ±0.1% of the specified amount.

PLANT—The term “plant” is intended to encompass plants at any stage ofmaturity or development, including a plant that has been detasseled orfrom which seed or grain have been removed. A seed or embryo that willproduce the plant is also included within the term plant.

PLANT PART—As used herein, the term “plant part” includes but is notlimited to pollen, tassels, seeds, branches, fruit, kernels, ears, cobs,husks, stalks, root tips, anthers, stems, roots, flowers, ovules,stamens, leaves, embryos, meristematic regions, callus tissue, anthercultures, gametophytes, sporophytes, microspores, protoplasts, and thelike. Tissue culture of various tissues of plants and regeneration ofplants therefrom is well known in the art. Plant cell as used hereinincludes plant cells that are intact in plants and/or parts of plants,plant protoplasts, plant tissues, plant cell tissue cultures, plantcalli, plant clumps, and the like. Further, as used herein, “plant cell”refers to a structural and physiological unit of the plant, whichcomprises a cell wall and also may refer to a protoplast. A plant cellof the present invention can be in the form of an isolated single cellor can be a cultured cell or can be a part of a higher-organized unitsuch as, for example, a plant tissue or a plant organ. Thus, as usedherein, a “plant cell” includes, but is not limited to, a protoplast, agamete producing cell, and a cell that regenerates into a whole plant.

ALLELE—Any alternative forms of sequence. Diploid cells carry twoalleles of the genetic sequence. These two sequence alleles correspondto the same locus (i.e., position) on homologous chromosomes.

ELITE INBRED, ELITE LINE—Maize plant that is substantially homozygousand which contributes useful agronomic and/or phenotypic qualities whenused to produce hybrids that are commercially acceptable.

GENE SILENCING—The loss or inhibition of the expression of a gene.

GENOTYPE—genetic makeup.

LINKAGE—The tendency of a segment of DNA on the same chromosome to notseparate during meiosis of homologous chromosomes. Thus during meiosisthis segment of DNA remains unbroken more often than expected by chance.

LINKAGE DISEQUILIBRIUM—The tendency of alleles to remain in linkedgroups when segregating from parents to progeny more often than expectedfrom chance.

LOCUS—A defined segment of DNA. This segment is often associated with anallele position on a chromosome.

PHENOTYPE—The detectable characteristics of a maize plant. Thesecharacteristics often are manifestations of the genotype/environmentinteraction.

BACKCROSS and BACKCROSSING refer to the process whereby a progeny plantis repeatedly crossed back to one of its parents. In a backcrossingscheme, the “donor” parent refers to the parental plant with the desiredgene or locus to be introduced. The “recipient” parent (used one or moretimes) or “recurrent” parent (used two or more times) refers to theparental plant into which the gene or locus is being introduced. Forexample, see Ragot, M. et al. Marker-assisted Backcrossing: A PracticalExample, in Techniques et Utilisations des Marqueurs Moleculaires LesColloques, Vol. 72, pp. 45-56 (1995); and Openshaw et al.,Marker-assisted Selection in Backcross Breeding, in Proceedings of theSymposium “Analysis of Molecular Marker Data,” pp. 41-43 (1994). Theinitial cross gives rise to the F1 generation. The term “BC1” refers tothe second use of the recurrent parent, “BC2” refers to the third use ofthe recurrent parent, and so on.

CROSS or CROSSED refer to the fusion of gametes via pollination toproduce progeny (e.g., cells, seeds or plants). The term encompassesboth sexual crosses (the pollination of one plant by another) andselfing (self-pollination, e.g., when the pollen and ovule are from thesame plant) and use of haploid inducer to form haploid seeds. The term“crossing” refers to the act of using gametes via pollination to produceprogeny.

CULTIVAR and VARIETY refer to a group of similar plants that bystructural or genetic features and/or performance can be distinguishedfrom other varieties within the same species.

TRANSGENE refers to any nucleotide sequence used in the transformationof a plant (e.g., maize), animal, or other organism. Thus, a transgenecan be a coding sequence, a non-coding sequence, a cDNA, a gene orfragment or portion thereof, a genomic sequence, a regulatory elementand the like. A “transgenic” organism, such as a transgenic plant, is anorganism into which a transgene has been delivered or introduced and thetransgene can be expressed in the transgenic organism to produce aproduct, the presence of which can impart an effect and/or a phenotypein the organism.

INTRODUCE OR INTRODUCING (and grammatical equivalents thereof) in thecontext of a plant cell, plant and/or plant part means contacting anucleic acid molecule with the plant, plant part, and/or plant cell insuch a manner that the nucleic acid molecule gains access to theinterior of the plant cell and/or a cell of the plant and/or plant parti.e. transformation. It also refers to both the natural and artificialtransmission of a desired allele, transgene, or combination of desiredalleles of a genetic locus or genetic loci, or combination of desiredtransgenes from one genetic background to another. For example, adesired allele or transgene at a specified locus can be transmitted toat least one progeny via a sexual cross between two parents of the samespecies, where at least one of the parents has the desired allele ortransgene in its genome. Alternatively, for example, transmission of anallele or transgene can occur by recombination between two donorgenomes, e.g., in a fused protoplast, where at least one of the donorprotoplasts has the desired allele in its genome. The desired allele maybe a selected allele of a marker, a QTL, a transgene, or the like.Offspring comprising the desired allele or transgene can be repeatedlybackcrossed to a line having a desired genetic background and selectedfor the desired allele or transgene, with the result being that thedesired allele or transgene becomes fixed in the desired geneticbackground.

I. Embodiments of the Invention

A. Inbred and Hybrid Production

Certain regions of the Corn Belt can have specific difficulties relatedto grain production that other regions may not have. Thus, the cornhybrids developed from inbreds should have traits that overcome or atleast minimize these regional growing problems. Examples of theseproblems include Gray Leaf Spot infection in the eastern Corn Belt, cooltemperatures during seedling emergence in the northern Corn Belt, CornLethal Necrosis (CLN) disease in the Nebraska region and soil withexcessively high pH levels in the west. Hybrid combinations employinbreds that address these specific issues resulting in the developmentof hybrids which are well adapted to niche production challenges.However, the aim of seed producers is to provide a number of traits toeach inbred so that the corresponding hybrid combinations can be usefulacross broad regions of the Corn Belt. Biotechnology techniques offertools, such as microsatellites, SNPs, RFLPs, RAPDs and the like, tobreeders to accomplish the goal of providing desirable traits ininbreds.

To produce hybrids, inbreds are developed using numerous methods, whichallow for the introduction of needed traits into the inbreds used in thehybrid combination. Hybrids are not often uniformly adapted for usethroughout the entire U.S. Corn Belt, but most often are adapted forspecific regions of the Corn Belts because for example, northern regionsof the Corn Belt require shorter season hybrids than do southernregions. Hybrids that grow well in Colorado and Nebraska soils may notflourish in richer Illinois and Iowa soils. Thus, several differentmajor agronomic traits are important in hybrid combination for growth inthe various Corn Belt regions, and these traits have an impact on hybridperformance.

If there is a pool of desirable maize varieties for use as parents thendevelopment of a corn hybrid involves one step crossing the selectedmaize variety with at least one different maize variety to produce thehybrid progeny. This single crossing step is possible because breedershave been developing inbreds from different maize germplasm pools sincethe early 1900s, which can be used in hybrid combinations. However, tokeep producing better and higher yielding hybrids, better inbreds mustbe developed. Inbred development involves the step of selecting plantsfrom various germplasm pools, or from the same germplasm pool for makinginitial breeding crosses; and then either producing haploid seed fromthe cross and selfing as needed, or selfing the breeding crosses forseveral generations to produce a series of inbred lines, which, althoughdifferent from each other, breed true and are highly uniform. Duringplant selection in each generation, uniformity of plant type ismaintained to ensure homozygosity and phenotypic stability. Aconsequence of the homozygosity and homogeneity of the inbred lines isthat the hybrid between a defined pair of inbreds, regardless of themethod by which the inbreds were produced, will always be the same.

The maize variety and seed of the present invention can be employed tocarry an agronomic package of this invention into a hybrid.Additionally, as described herein the inbred line can comprise one ormore transgenes that are then introduced into the hybrid seed. When themaize variety parents that give a superior hybrid have been identified,the hybrid seed can be reproduced indefinitely as long as thehomogeneity of the maize variety parents is maintained.

Any breeding methods using the maize variety HAF4561, and its progenyare part of this invention. Inbred development can be accomplished bydifferent methods, for example, pedigree selection, backcrossing,recurrent selection, haploid/doubled haploid production. An inbred plantwith similar genetic or characteristics to maize variety HAF4561 couldbe produced by applying double haploid methods to the progeny of a crossbetween maize inbred HAF4561 and a different plant. Double haploidmethods produce substantially homozygous plants without repeatedbackcrossing steps. The haploid/doubled haploid process of developinginbreds starts with the induction of a haploid by using, for example,KWS inducers lines, Krasnador inducers lines, stock six inducer lines(Coe, 1959, Am. Nat. 93:381-382). The haploid cell is then doubled, andthe doubled haploid plant is produced. In some embodiments, a method ofproducing an maize plant with doubled haploid chromosomes derived fromthe maize variety HAF4561 the method comprising: (a) crossing a plant,wherein said plant may have one or more traits, with an inducer maizeplant to produce a progeny with haploid chromosomes; and (b) doublingthe haploid chromosomes in the progeny to produce a maize plant withdoubled haploid chromosomes. In some embodiments, the progeny may be forexample a cell, seed, embryo or plant. In further embodiments, the maizeplant with doubled haploid chromosomes produced by step (b) above is amaize inbred plant with the characteristics of maize variety HAF4561. Inother embodiments, the plant crossed with an inducer in step (a) is ahybrid maize plant produced by crossing maize variety HAF4561 with adifferent plant.

For examples of the use of double hybrid methods, see Prasanna et al.(eds) Doubled Haploid Technology in Maize Breeding: Theory and PracticeMexico, D. F.: CIMMYT, Barnabus et al. “Colchicine, an efficient genomedoubling agent for maize microsporescultured in anthero”, Plant CellReports, v. 18:858-862, 1999 or US patent application 2003/0005479.Sometimes this doubled haploid can be used as an inbred but sometimes itis further self pollinated to finish the inbred development. Anotherbreeding process is pedigree selection which uses the selection in an F2population produced from a cross of two genotypes (often elite inbredlines), or selection of progeny of synthetic varieties, open pollinated,composite, or backcrossed populations. Pedigree selection is effectivefor highly heritable traits but other traits, such as yield, requirereplicated test crosses at a variety of stages for accurate selection.

The maize variety and hybrid corn lines of the present invention can beemployed in a variety of breeding methods that can be selected,depending on the mode of reproduction, the trait and/or the condition ofthe germplasm. Thus, any breeding methods using the inbred corn lineHAF4561 or it progeny are part of this invention. Such methods caninclude, but are not limited to, marker assisted breeding, selection,selfing, backcrossing, hybrid production, and crosses to populations.

All plants and plant cells produced using maize variety HAF4561 areencompassed within the present invention, which also encompasses thecorn variety used in crosses with other, different, corn varieties toproduce corn hybrid seeds and hybrid plants and the grain produced onthe hybrid plant. This invention includes progeny plants and plantcells, which upon growth and differentiation produce corn plants havingthe physiological and morphological characteristics of the maize varietyHAF4561 when grown in the same environmental conditions.

Maize breeders select for a variety of traits in inbred plants thatimpact hybrid performance in addition to selecting for acceptableparental traits. Such traits include, but are not limited, to yieldpotential in hybrid combination, dry down, maturity, grain moisture atharvest, green snap, resistance to root lodging, resistance to stalklodging, grain quality, disease and insect resistance, ear, and plantheight. Additionally, because hybrid performance may differ in differentsoil types such as those having low levels of organic matter, clay,sand, black, high pH, or low pH; or in different environments such aswet environments, drought environments, and no tillage conditionsmultiple trials testing for agronomic traits must be run to asserthybrid performance across environments. These traits are governed by acomplex genetic system that makes selection and breeding of an inbredline extremely difficult. However, even if an inbred, in hybridcombination, has excellent yield (a desired characteristic), it may notbe useful for hybrid seed production if the inbred lacks acceptableparental traits, for example, seed size, pollen production, good silks,plant height, etc.

The following example is provided to illustrate the difficulty ofbreeding and developing inbred lines. Two inbreds compared forsimilarity of 29 traits differed significantly for 18 traits between thetwo lines. If 18 simply inherited single gene traits were polymorphicwith gene frequencies of 0.5 in the parental lines, and assumingindependent segregation (as would essentially be the case if each traitresided on a different chromosome arm), then the specific combination ofthese traits as embodied in an inbred would only be expected to becomefixed at a rate of one in 262,144 possible homozygous geneticcombinations. Selection of the specific inbred combination is alsoinfluenced by the specific selection environment on many of these 18traits which makes the probability of obtaining this one inbred evenmore remote. In addition, most traits in the corn genome are not singledominant genes; they are multi-genetic with additive gene action but notdominant gene action. Thus, the general approach of producing anon-segregating F1 generation and self pollinating to produce an F2generation that segregates for traits and then selecting progeny fromthe F2 generation with the desired visual traits does not easily lead toa useful inbred. Great care and breeder expertise must be used in theselection of breeding material to continue to increase yield and enhancedesirable agronomic features of inbreds and resultant commercialhybrids.

In one embodiment, a method of producing a plant of this invention is byplanting the seed of HAF4561, which is substantially homozygous,self-pollinating or sib pollinating the resultant plant in isolateenvironment, and harvesting the resultant seed. The F1 hybrid seed canbe produced using two distinct inbreds, the male inbred contributingpollen to the female seed producing parent, the female seed producingparent, on the other hand, is not contributing pollen to the seed. Thus,in some embodiments, a method is provided for producing an hybrid maizeseed by crossing a plant of maize variety HAF4561 with a different maizeplant (e.g., a different inbred line), and harvesting the resultanthybrid maize seed. A maize plant of the present invention can act as amale or female part in hybrid production.

A method is also provided for producing maize seed comprising growingthe plant of this invention until seed is produced and harvesting theseed, wherein the harvested seed is inbred or hybrid or haploid seed.Plants and plant parts produced by the seed of this method is alsoprovided herein. Additionally, provided herein is a method of producinghybrid seed corn from this inbred corn line and producing hybrid plantsand seeds from the hybrid seed corn of this invention.

Thus, in some embodiments, the invention provides hybrid seed, producedby planting, in pollinating proximity, seeds of corn inbred line HAF4561and seeds of another inbred line. The corn plants resulting from saidplanting are cultivated; emasculation of one of the inbred lines (i.e.,the selected inbred plant) and allowing pollination to occur. Seedsproduced by plants of the selected inbred can be harvested. In furtherembodiments, seeds of corn inbred line HAF4561 are planted andcultivated. Alternatively, emasculated plants are pollinated withpreserved maize pollen (as described in U.S. Pat. No. 5,596,838 toGreaves). The seeds produced by the inbred line HAF4561 pollinated withthe preserved pollen can be harvested. The hybrid seed produced by thehybrid combination of plants of inbred corn seed designated HAF4561 andplants of another inbred line or produced by the plants of inbred cornseed designated HAF4561 pollinated by preserved pollen are included inthe present invention. This invention further encompasses hybrid plantsand plant parts thereof including but not limited to the grain andpollen of the plant grown from this hybrid seed.

In two alternative embodiments, the method is provided for producing anhybrid maize seed, the method comprising crossing a plant of maizevariety plant HAF4561 with a different maize variety (e.g., a differentinbred line), wherein the pollen of the maize variety HAF4561 pollinatesthe different maize variety, or in the alternative the pollen of thedifferent maize variety pollinates maize variety HAF4561, and theresultant hybrid maize seed is harvested.

In particular embodiments, this invention is directed to the uniquecombination of traits that combine in corn line HAF4561. Alsoencompassed within this invention is an F1 hybrid maize seed comprisingan inbred maize plant cell of inbred maize plant HAF4561.

The invention further relates to methods for producing other maizebreeding lines derived from the corn inbred of this invention bycrossing the maize inbred plant HAF4561 with a second maize plant andgrowing the progeny seed to yield a inbred HAF4561-derived maize plant.Thus, in some embodiments of this invention, a method is provided forproducing a maize plant derived from the inbred plant HAF4561, themethod comprising the steps of: (a) growing a hybrid progeny plantwherein the maize variety of this invention is a parent (b) crossing thehybrid progeny plant with itself or a different plant to produce a seedof a progeny plant; (c) growing the progeny plant from said seed andcrossing the progeny plant with itself or a different plant; and (d)repeating steps (c) for an additional generation to produce a maizeplant derived from the inbred plant HAF4561. The present invention alsoprovides a maize seed produced by crossing the plant of this inventionwith itself or a different maize plant.

Thus, other aspects of this invention include a method for developing amaize plant in a maize plant breeding program, comprising applying plantbreeding techniques comprising recurrent selection, backcrossing,pedigree breeding, marker enhanced selection, haploid/double haploidproduction, or transformation to the maize plant of this invention, orits parts, wherein application of said techniques results in developmentof a maize plant.

B. Transfer of Additional Traits into Inbred Corn Line HAF4561

A specific location on a chromosome can be referred to as a locus. Traitconversion refers to a variety that has been modified such that thevariety retains its physiological and morphological characteristicsexcept for those changed by the introduction of the trait. Thus avariety undergoing a herbicide resistance trait conversion will evidencethe additional trait of resisting damage by the herbicide. The varietywill after trait conversion have one or more loci with a specificdesired trait. Such a variety modification may be through mutant genes,transgenes, or native traits. A maize line and any minor geneticmodifications which may include a trait conversion, a mutation, or avariant is a variety.

The use of an inbred maize plant, such as the inbred of the presentinvention, as a recurrent parent in a breeding program is referred to asbackcrossing. Backcrossing is often employed to introduce an additionaltrait (e.g., targeted trait or trait of interest) or trait(s), eithertransgenic or nontransgenic, into a recurrent parent. A plant with adesired trait or locus is crossed into a recurrent maize parent usuallyin one or more backcrosses. If markers are employed to assist inselection of progeny that have the desired trait and recurrent parentbackground genetics, then the number of backcrosses needed to recoverthe recurrent parent with the desired trait or locus can be relativelyfew, e.g., two or three. However, 3, 4, 5 or more backcrosses are oftenrequired to produce the desired inbred with the gene or locus conversionin place. The number of backcrosses needed for a trait introduction isoften linked to the genetics of the line carrying the trait and therecurrent parent and the genetics of the trait. Multigenic traits,recessive alleles and unlinked traits can affect the number ofbackcrosses that may be necessary to achieve the desired backcrossconversion of the inbred.

Basic maize crossing techniques, as well as other corn breeding methodsincluding recurrent, bulk or mass selection, pedigree breeding, openpollination breeding, marker assisted selection/breeding, doublehaploids development and selection breeding are well known in the art(see, e.g., Hallauer, Corn and Corn Improvement, Sprague and Dudley, 3rdEd. 1998). Dominant, single gene traits or traits with obviousphenotypic changes are particularly well managed in backcrossingprograms, as are well known in the art. A backcross conversion or locusconversion both refer to a product of a backcrossing program.

A backcrossing program is more complicated when the trait is a recessivegene. A determination of the presence of the recessive gene requires theuse of some testing to determine if the trait has been transferred. Useof markers to detect the gene reduces the complexity of traitidentification in the progeny. A marker specific for a recessive trait,such as a single nucleotide polymorphism (SNP), can increase theefficiency and speed of tracking the recessive trait within abackcrossing program.

The last backcross generation can be selfed, if necessary, to give purebreeding progeny for the nucleic acid(s) being transferred. Theresulting plants generally have essentially all of the morphological andphysiological characteristics of the inbred corn line of interest, inaddition to the transferred trait(s) (e.g., one or more gene traits).The exact backcrossing protocol will depend on the trait being alteredto determine an appropriate testing protocol.

Thus, in some embodiments, one or more additional traits can beintroduced into a plant of this invention using any method known in theart for introducing traits into plants. Nucleotide sequences encodingtraits of interest can all be located at the same genomic locus in thedonor, non-recurrent parent, and in the case of transgenes, can be partof a single DNA construct integrated into the donor's genome or intoadditional chromosomes integrated into the donor's genome.Alternatively, if the nucleotide sequences of interest are located atdifferent genomic loci in the donor, non-recurrent parent, backcrossingcan be carried out to establish all of the morphological andphysiological characteristics of the plant of the invention in additionto the nucleotide sequences encoding the traits of interest in theresulting maize inbred line.

Accordingly, the present invention provides a method of introducing orintrogressing at least one additional trait into the maize inbred lineHAF4561, comprising the steps of: (a) crossing a plant grown from theseed of the maize inbred line HAF4561 (which is the recurrent parent,representative seed of which has been deposited), with a donor plant ofanother maize line that comprises at least one additional trait toproduce F1 plants; (b) selecting F1 plants having the at least oneadditional trait to produce the selected F1 progeny plants; (c) crossingthe F1 plants of (b) with the recurrent parent to produce backcrossedprogeny plants having the at least one additional trait; (d) selectingfor backcrossed progeny plants that have at least one of the additionaltraits and physiological and morphological characteristics of maizeinbred line of the recurrent parent to produce selected backcrossedprogeny plants; and (e) repeating the crossing of the selectedbackcrossed progeny to the recurrent parent of step (c) and theselecting of step (d) in succession to produce a plant that comprises atleast one additional trait and all of the physiological andmorphological characteristics of the maize inbred line HAF4561 whengrown in the same environmental conditions (e.g., essentially therecurrent parent having the at least one additional trait).

In some embodiments of this invention, the at least one additional traitcomprises the trait of male sterility, herbicide resistance, insectresistance, disease resistance, altered starch, modified amylase starch,amylose starch, waxy starch, or any combination thereof. In otherembodiments of this invention, the at least one desired trait isconferred by a nucleic acid molecule encoding an enzyme that includes,but is not limited to, a phytase, a stearyl-ACP desaturase, afructosyltransferase, a levansucrase, an amylase, an invertase, a starchbranching enzyme, or any combination thereof.

In some embodiments, the selecting and crossing steps of (e) arerepeated at least 3 times in order to produce a plant that comprises theat least one desired trait and all of the physiological andmorphological characteristics of the maize inbred line of the recurrentparent in the present invention (listed in Table 1) when grown under thesame environmental conditions (as determined at the 5% significancelevel). In other embodiments, the selecting and crossing steps of (e)are repeated from 0 to 2 times, from 0 to 3 times, from 0 to 4 times, 0to 5 times, from 0 to 6 times, from 0 to 7 times, from 0 to 8 times,from 0 to 9 times or from 0 to 10 times, in order to produce a plantthat comprises the at least one additional trait and all of thephysiological and morphological characteristics of the maize inbred lineof the recurrent parent in the present invention. In other embodiments,the crossing and growing steps of (a) and (b) in step (c) are repeatedfrom 0 to n times (wherein n can be any number) in order to produce aplant that comprises the at least one additional trait and all of thephysiological and morphological characteristics of the maize inbred lineof the recurrent parent in the present invention.

The method of introducing traits as described herein can be done withfewer back crossing events if the trait and/or the genotype of thepresent invention is selected for or identified through the use ofmarkers. SSR, microsatellites, single nucleotide polymorphisms (SNPs)and the like decrease the amount of breeding time required to locate aline with the desired trait or traits and the characteristics of thepresent invention. Backcrossing in two or even three traits (for examplethe glyphosate resistance, Europe corn borer resistance, corn rootwormresistance) is routinely done with the use of marker assisted breedingtechniques and or selection pressure testing. Introduction of transgenesor mutations into a maize line is known as single gene conversion. Morethan one gene and, in particular, transgenes and/or mutations that arereadily tracked with markers, can be moved during the same “single geneconversion” process. This single gene conversion process results in aline comprising more desired or targeted traits than just the one butstill having the characteristics of the plant line of the presentinvention plus those characteristics added by the desired/targetedtraits.

Genetic variants of inbred corn line HAF4561 that arenaturally-occurring or created through traditional breeding methodsusing inbred corn line HAF4561 are also intended to be within the scopeof this invention. In particular embodiments, the invention encompassesplants of this invention and parts thereof further comprising one ormore additional traits, in particular, specific, single gene transferredtraits. Examples of traits that may be transferred include, but are notlimited to, herbicide resistance, disease resistance (e.g., bacterial,fungal or viral disease), nematode resistance, tolerance to abioticstresses (e.g., drought, temperature, salinity), yield enhancement,improved nutritional quality (e.g., oil starch and protein content orquality), modified metabolism (e.g. protein, carbohydrates, starch,amylase,) altered reproductive capability (e.g., male sterility) orother agronomically important traits.

Such traits may be introduced into a plant of this invention fromanother corn line or through direct transformed into a plant of thisinvention (discussed below). One or more new traits can be transferredto a plant of this invention, or, alternatively, one or more traits of aplant of this invention are altered or substituted. The introduction ofthe trait(s) into a plant of this invention may be achieved by anymethod of plant breeding known in the art, for example, pedigreebreeding, backcrossing, doubled-haploid breeding, and the like.

C. Nucleic Acids for Introduction into Maize Plants of the PresentInvention

As would be appreciated by one of skill in the art, any nucleotidesequence of interest can be introduced into the plants and/or partsthereof of the present invention. Some exemplary nucleotide sequencesand traits that may be used with the present invention are providedherein.

Methods and techniques for introducing and/or introgressing a trait ornucleotide sequence into a plant of the present invention throughbreeding, transformation, site specific insertion, mutation and thelike, are well known and understood by those of ordinary skill in theart. Nonlimiting examples of such techniques include, but are notlimited to, anther culturing, haploid/double haploid production,(including, but not limited to, stock six, which is a breeding/selectionmethod using color markers), transformation, irradiation to producemutations, and chemical or biological mutation agents.

1. Male Sterility

As described herein, the inbred and hybrid lines plants of thisinvention can comprise male sterility. Male sterility and/or CMS(cytoplasmic male sterility) systems for maize parallel the CMS typesystems, were first used in maize in the seventies but were to widelyembraced; however, CMS has have been routinely used in hybrid productionin sunflower plants. A number of methods are available to generate malesterile plants including, but not limited to, introduction into theplant of nucleotide sequences that confer male sterility, by chemicals,and/or by a mixture of nucleotide sequences conferring male sterility,natural or induced sterility mutations, and/or chemicals.

As described herein, the inbred and hybrid plants of this invention cancomprise the trait of male sterility. Male sterility is useful, forexample, in hybrid production for elimination of pollen shed from theseed producing parent. Sterility can be produced by pulling or cuttingtassels from the plant, i.e., detasseling, use of gametocides, or use ofgenetic material to render the plant sterile using a CMS type of geneticcontrol or a nuclear genetic sterility, use of chemicals, for exampleherbicides that inhibit or kill pollen. The seed producing parent can begrown in isolation from other pollen sources except for the pollensource which is the male fertile inbred, which serves as the male parentin the hybrid. To facilitate pollination of the seed producing (female)parent, the male fertile inbreds can be planted in rows near the malesterile (female) inbred.

In hybrid seed production using the standard CMS system, three differentmaize lines are employed. The first line is cytoplasmic male-sterile.This line will be the seed producing parent line. The second line is afertile inbred that is the same as or isogenic with the seed producinginbred parent but lacking the trait of male sterility. This is amaintainer line used to make new inbred seed of the seed producing malesterile parent. The third line is a different inbred which is fertile,has normal cytoplasm and carries a fertility restoring gene. This lineis called the restorer line in the CMS system. The CMS cytoplasm isinherited from the maternal parent (or the seed producing plant);therefore in order for the hybrid seed produced on such a plant to befertile, the pollen used to fertilize this plant must carry the restorergene. The positive aspect of this process is that it allows hybrid seedto be produced without the need for detasseling the seed parent.However, this system does require breeding of all three types oflines: 1) a male sterile line-to carry the CMS, 2) a maintainer line;and 3) a line carrying the fertility restorer gene.

Accordingly, in some embodiments of the present invention, sterilehybrids are produced and the pollen necessary for the formation of grainon these hybrids is supplied by interplanting of fertile inbreds in thefield with the sterile hybrids.

A number of additional techniques exist that are designed to avoiddetasseling in maize hybrid production. Nonlimiting examples of suchtechniques include switchable male sterility, lethal genes in the pollenor anther, inducible male sterility and/or male sterility genes withchemical restorers. Additional examples include, but are not limited to,U.S. Pat. No. 6,025,546, which describes the use of tapetum-specificpromoters and the barnase gene to produce male sterility, and U.S. Pat.No. 6,627,799, which describes modifying stamen cells to provide malesterility. Therefore, one aspect of the present invention provides acorn plant of this invention comprising one or more nucleotide sequencesthat restore male fertility to male-sterile maize inbreds or hybridsand/or one or more nucleotide sequences or traits to produce malesterility in maize inbreds or hybrids.

Furthermore, methods for genetic male sterility are disclosed in EPOPublication No. 89/3010153.8, PCT Publication No. WO 90/08828 and U.S.Pat. Nos. 4,654,465, 4,727,219, 3,861,709, 5,432,068 and 3,710,511.Gametocides, some of which are taught in U.S. Pat. No. 4,735,649(incorporated by reference) can be employed to make the plant malesterile. Gametocides, including, but not limited to, glyphosate, and itsderivatives are chemicals or substances that negatively affect thepollen or at least the fertility of the pollen and provide malesterility to the seed producing parent.

It is noted that hybrid production employing any most forms of malesterility including mechanical emasculation can have a small occurrenceof self pollinated female inbred seeds along with the intended F1 hybridseeds. Great measures are taken to avoid the inbred seed production in ahybrid seed production field; but inbred seed can occur during F1 seedproduction and it gets harvested with the hybrid seed harvest.

Inbred seed in a sample of hybrid seed may be detected using molecularmarkers. Alternatively, the seed sample can be planted and an inbredcapture process can be used to isolate inbred seed from the hybrid F1seed sources. The inbred plants tend to be readily distinguished fromthe hybrid plants due to the inbreds having a stunted appearance, i.e.,shorter plant, smaller ear, etc. Self pollination of the stunted plantsgrown from these identified putative inbred plants produces either thefemale inbred seed, if it was an inbred plant or if it was a weak hybridthan the hybrid kernel will be F2 seed. The resultant plants areobserved for size or they can be tested by markers to identify anyinbred plants. The identified inbred plants can be selected andself-pollinated to form the inbred seed.

2. Additional Traits of Interest

As discussed above, backcrossing of recessive traits has allowed knownmutant traits to be moved into elite germplasm. Mutations can beintroduced in germplasm by the plant breeder. Mutations can also resultfrom plant or seed or pollen exposure to temperature alterations,culturing, radiation in various forms, chemical mutagens like EMS andlike, as are well known in the art. Non-limiting examples of mutantgenes that have been identified and introduced into elite maize usefulwith this invention include the genotypes numerous sterility and partialsterility genes, herbicide resistant mutants, phytic acid mutants, waxy(wx), amylose extender (ae), dull (du), horny (h), shrunken (sh),brittle (bt), floury (fl), opaque (O), and sugary (su). Some of thebracketed nomenclature for these mutant genes is based on the effectthese mutant genes have on the physical appearance and phenotype of thekernel.

Additional mutations useful with this invention include, but are notlimited to, those that result in the production of starch with markedlydifferent functional properties even though the phenotypes of the seedand plant remain the same. Such genotypes include, but are not limitedto, sugary-1 (su1), sugary-2 (su2); shrunken 1 (sh1) and shrunken 2(sh2).

Additional, exemplary nucleic acid molecules that can be introduced intoa plant of the present invention include, but are not limited to,nucleotide sequences that confer insect resistance including, but notlimited to, resistance to Corn Rootworm in the event DAS-59122-7, Mir604Modified Cry3A event, Event 5307 Syngenta, MON 89034, MON 88017 Bacillusthuringiensis (Cry genes) Cry34/35Ab1, Cry1A.105, Cry1F, Cry2Ab2, Cry1A,Cry1AB, Cry1Ac Cry3Bb1, or any combination thereof. Thus, for example,in some embodiments, an insecticidal gene that can be introduced into aplant of the present invention is a Cry1Ab gene or a portion thereof,for example, introduced into a plant of the present invention from amaize line comprising a Bt-11 event as described in U.S. Pat. No.6,114,608, (incorporated herein by reference) or from a maize linecomprising a 176 Bt event as described in Koziel et al. (Biotechnology11: 194-200 (1993)).

In other embodiments of this invention, nucleotide sequences that conferdisease resistance are introduced and/or transformed into the inbredline. Non-limiting examples of such nucleotide sequences include, butare not limited to, a nucleotide sequence encoding Mosaic virusresistance, a nucleotide sequence encoding an MDMV strain B coat proteinwhose expression confers resistance to mixed infections of maize dwarfmosaic virus and maize chlorotic mottle virus (Murry et al.Biotechnology (1993) 11:1559-64, a nucleotide sequence conferringresistance to Northern corn leaf blight, and a nucleotide sequenceconferring resistance to Southern corn leaf blight, or any combinationthereof.

In additional embodiments, nucleotide sequences that confer herbicideresistance/tolerance are useful with the present invention, non-limitingexamples of which comprise nucleotide sequences conferring resistance toherbicides for example imazethapyr, glyphosate, dicamba, and the like,and nucleotide sequences encoding Pat(phosphinothricin-N-acetyltransferase), Bar (bialophos), alteredacetohydroxyacid synthase (AHAS) (confers tolerance to variousimidazolinone or sulfonamide herbicides) (U.S. Pat. No. 4,761,373), orany combination thereof.

Additional, non-limiting examples of nucleotide sequences conferringherbicide resistance/tolerance that are useful with the presentinvention, include nucleotide sequences conferring tolerance toimidazolinones (e.g., a “IT” or “IR” trait). U.S. Pat. No. 4,975,374(incorporated herein by reference), relates to plant cells and plantscontaining a gene encoding a mutant glutamine synthetase (GS) havingresistance to inhibition by herbicides that are known to inhibit GS,e.g., phosphinothricin and methionine sulfoximine. Also, expression of aStreptomyces bar gene encoding a phosphinothricin acetyl transferase inmaize plants confers tolerance to the herbicide phosphinothricin orglufosinate (U.S. Pat. No. 5,489,520). U.S. Pat. No. 5,013,659,(incorporated herein by reference), is directed to plants that express amutant acetolactate synthase (ALS) that renders the plants resistant toinhibition by sulfonylurea herbicides. U.S. Pat. No. 5,162,602 disclosesnucleotide sequences that confer resistance to cyclohexanedione andaryloxyphenoxypropanoic acid herbicides. The tolerance is conferred byan altered acetyl coenzyme A carboxylase (ACCase). U.S. Pat. No.5,554,798 discloses transgenic glyphosate tolerant maize plants, whichtolerance is conferred by an altered 5-enolpyruvyl-3-phosphoshikimate(EPSP) synthase gene. U.S. Pat. No. 5,804,425 discloses transgenicglyphosate tolerant maize plants, which tolerance is conferred by anEPSP synthase gene derived from Agrobacterium tumefaciens CP-4 strain.Also, tolerance to a protoporphyrinogen oxidase inhibitor is achieved byexpression of a protoporphyrinogen oxidase enzyme in plants as disclosedin U.S. Pat. Nos. 5,767,373, 6,282,837, or WO 01/12825. Another traittransferable to the plant of the present invention confers a safetyeffect or additional tolerance to an inhibitor of the enzymehydroxyphenylpyruvate dioxygenase (HPPD) and transgenes conferring suchtrait are, for example, described in PCT Publication Nos. WO 9638567, WO9802562, WO 9923886, WO 9925842, WO 9749816, WO 9804685 and WO 9904021.Any of the above described nucleotide sequences identified to conferherbicide resistance/tolerance can be used to confer suchresistance/tolerance to the plants of the present invention. Thesenucleotide sequences can be introduced or transformed into the plants ofthe present invention alone or in any thereof.

Additional embodiments of this present invention include nucleotidesequences conferring altered traits. Such altered traits include, butare not limited to, lignin composition and production (including but notlimited to nucleotide sequences conferring the brown mid-rib trait),flowering, senescence, and the like, or any combination thereof.

The present invention also encompasses methods for the introduction intoa plant of this invention, one or more traits that have an effect onproducts or by-products of the corn plant such as the sugars, oils,protein, ethanol, biomass and the like. Such traits can include thosethat result in the formation of an altered carbohydrate or alteredstarch. An altered carbohydrate or altered starch can be formed as aresult of expression of one or more introduced nucleotide sequences thataffect synthases, branching enzymes, pullanases, debranching enzymes,isoamylases, alpha amylases, beta amylases, AGP, ADP and other enzymeswhich affect amylose and/or amylopectin ratio or content, or thebranching pattern of starch.

Introduced fatty acid modifying nucleotide sequences can also affectstarch content and therefore can be employed in the methods and plantsof this invention. Additionally, introduced nucleotide sequences thatare associated with or affect starch and carbohydrates can be adapted sothat the nucleotide sequence or its enzyme product does not necessarilyalter the form or formation of the starch or carbohydrate of the seed orplant but instead the introduced nucleotide sequence or its RNA,polypeptide, protein or enzyme can be adapted to degrade, alter, orotherwise change the formed starch or carbohydrate. Examples of thistechnology are shown, for example, in U.S. Pat. Nos. 7,033,627,5,714,474, 5,543,570, 5,705,375, 7,102,057, each of which areincorporated by reference. An example of the use of an alpha amylaseadapted in this manner in maize is described in U.S. Pat. No. 7,407,677,the content of which is also incorporated herein by reference.

By way of example only, specific events (followed by their APHISpetition numbers) that can be introduced into maize plants by backcrossbreeding techniques include Monsanto's Drought Tolerant/MON87460(09-055-01p); Male Sterile, Fertility Restored, VisualMarker/DP-32138-1 (08-338-01p); (07-253-01p) Syngenta LepidopteranResistant/MIR 162; (07-152-01p) Pioneer's Corn Glyphosate &Imidazolinone Tolerant/98140; (04-362-01p) Syngenta Corn Corn RootwormProtected/MIR604; (04-229-01p) Monsanto Corn High Lysine/LY038;04-125-01p Monsanto Corn Corn Rootworm Resistant/MON 88017; 03-353-01pDow Corn Corn Rootworm Resistant/59122; (03-181-01p) Dow CornLepidopteran Resistant & Phosphinothricin Tolerant/6275; (01-137-01p)Monsanto Corn Corn Rootworm Resistant/MON 863; (00-136-01p) Mycogen doDow & Pioneer Corn Lepidopteran Resistant PhosphinothricinTolerant/1507; (97-099-01p) Monsanto Corn Glyphosate Tolerant/NK603;(98-349-01p) AgrEvo Corn Phosphinothricin Tolerant and Male Sterile/MS6;(97-342-01p) Pioneer Corn Male Sterile and PhosphinothricinTolerant/676, 678, 680; (97-265-01p) AgrEvo Corn PhosphinothricinTolerant and Lepidopteran Resistant/CBH-351; (96-017-01p) Monsanto CornEuropean Corn Borer Resistant/MON 809, MON 810; (09-233-01p) Dow Corn2,4-D and ACCase-Inhibitor Tolerant/DAS-40278-9; (11-244-01p) PioneerCorn Insect Resistant and Glufosinate Tolerant/DP-ØØ4114-3; (10-336-01p)Syngenta Corn Rootworm Resistant/5307; (10-281-01p) Monsanto Corn MaleSterile/MON 87427; (11-342-01p) Genective Corn GlyphosateTolerant/VCO-Ø1981-5; glyphosate tolerant event GA21 (97-09901p), theglyphosate tolerant event NK603 (00-011-01p), the glyphosatetolerant/Lepidopteran insect resistant event MON 802 (96-31701p) Mon810,the Lepidopteran insect resistant event DBT418 (96-29101p), the malesterile event MS3 (95-22801p), the Lepidopteran insect resistant eventBt11 (95-19501p), the phosphinothricin tolerant event B16 (95-14501p),the Lepidopteran insect resistant events MON 80100 (95-09301p) and MON863 (01-137-01p), the phosphinothricin tolerant events T14, T25(94-35701p), the Lepidopteran insect resistant event 176 (94-31901p),Western corn rootworm (04-362-01p), the phosphinothricin tolerant andLepidopteran insect resistant event CBH-351 (92-265-01p), the transgeniccorn event designated 3272 (05-280-01p) Syngenta's Corn ThermostableAlpha-amylase/3272 as described in US Patent Publication No. 20060230473(hereby incorporated by reference) and the like, or any combinationthereof. Additionally, the genes, promoters, transit peptides, targetingsequence and other genetic material used to form these varioustransgenic events can be used in to form a new transgene, promoter,targeting, etc. or to configure a synthetic gene for use intransformation, and after this genetic material is transformed into aline, which is stable. The new event can be backcrossed into germplasmof the present invention.

In some embodiments, a combination of traits can be transformed orintroduced into the plants of the present invention. This in someembodiments, a transgene can be introduced into a plant of a presentinvention which comprises a nucleotide sequence conferring tolerance toa herbicide and at least another nucleotide sequence encoding anothertrait, such as for example, an insecticidal protein. Such a combinationof single traits can be, for example, a Cry1Ab gene and a bar gene. Theintroduction of a Bt11 event into a maize line, such as the line of thepresent invention, by backcrossing is exemplified in U.S. Pat. No.6,114,608, and the present invention includes methods of introducing aBt11 event into a plant of the present invention and to progeny thereofusing, e.g., markers as described in U.S. Pat. No. 6,114,608.

D. Transformation of Corn Inbred HAF4561 Plants and/or Parts Thereof

The term transgenic plant refers to a plant having one or more geneticsequences that are introduced into the genome of a plant by atransformation method and the progeny thereof. With the advent ofmolecular biological techniques that have allowed the isolation andcharacterization of nucleic acids that encode specific protein products,scientists in the field of plant biology developed a strong interest inengineering the genome of plants to contain and express foreign nucleicacids, or additional, or modified versions of native or endogenousnucleic acids (perhaps driven by different promoters) in order to alterthe traits of a plant in a specific manner. Such foreign, additionaland/or modified nucleic acids are referred to herein collectively as“transgenes.” The term “transgene,” as used herein, is not necessarilyintended to indicate that the foreign nucleic acid is from a differentplant species. For example, the transgene may be a particular allelederived from another corn line or may be an additional copy of anendogenous gene. Over the last twenty to twenty-five years severalmethods for producing transgenic plants have been developed. Therefore,in particular embodiments, the present invention also encompassestransformed plants and/or parts thereof (e.g., cells, seeds, anthers,ovules, and the like) of inbred corn line HAF4561.

Transformation methods are techniques for integrating new nucleotidesequence(s) into the genome of a plant by recombinant nucleic acidtechnology, rather than by standard breeding practices. However, once atransgene is introduced into plant material and stably integrated,standard breeding practices can be used to move the transgene into othergermplasm.

Plant transformation generally involves the construction of anexpression vector that will function in plant cells. Such a vectorcomprises DNA or RNA comprising a nucleic acid under control of, oroperatively linked to, a regulatory element (for example, a promoter).The expression vector may contain one or more such operably linkednucleic acid/regulatory element combinations. The vector(s) may be inthe form of, for example, a plasmid or a virus, and can be used, aloneor in combination with other vectors, to provide transformed maizeplants, using transformation methods as described below to incorporatetransgenes into the genetic material of the maize plant(s).

Any transgene(s) known in the art may be introduced into a maize plant,tissue, cell or protoplast according to the present invention, e.g., toimprove commercial or agronomic traits, herbicide resistance, diseaseresistance (e.g., to a bacterial fungal or viral disease), insectresistance, nematode resistance, yield enhancement, nutritional quality(e.g., oil starch and protein content or quality), altered reproductivecapability (e.g., male sterility), and the like or any combinationthereof. Alternatively, a transgene may be introduced for the productionof recombinant proteins (e.g., enzymes) or metabolites.

A recombinant nucleic acid molecule of the invention can be introducedinto a plant cell in a number of art-recognized ways. Suitable methodsof transforming plant cells include microinjection (Crossway et al.BioTechniques 4:320-334 (1986)), electroporation (Riggs et al. Proc.Natl. Acad. Sci. USA 83:5602-5606 (1986)), Agrobacterium-mediatedtransformation (Hinchee et al. Biotechnology 6:915-921 (1988)), directgene transfer (Paszkowski et al. EMBO J. 3:2717-2722 (1984)), ballisticparticle acceleration using devices available, e.g., from Agracetus,Inc., Madison, Wis. and Dupont, Inc., Wilmington, Del. (see, forexample, Sanford et al., U.S. Pat. No. 4,945,050; and McCabe et al.Biotechnology 6:923-926 (1988)), protoplast transformation/regenerationmethods (see U.S. Pat. No. 5,350,689, issued Sep. 27, 1994 to Ciba-GeigyCorp.), Whiskers technology (See U.S. Pat. Nos. 5,464,765 and 5,302,523)and pollen transformation (see U.S. Pat. No. 5,629,183). See alsoWeissinger et al. Annual Rev. Genet. 22:421-477 (1988); Sanford et al.Particulate Science and Technology 5:27-37 (1987)(onion); Christou etal. Plant Physiol. 87:671-674 (1988)(soybean); McCabe et al.Bio/Technology 6:923-926 (1988)(soybean); Datta et al. Bio/Technology8:736-740 (1990)(rice); Klein et al. Proc. Natl. Acad. Sci. USA85:4305-4309 (1988)(maize); Klein et al. Bio/Technology 6:559-563(1988)(maize); Klein et al. Plant Physiol. 91:440-444 (1988)(maize);Fromm et al., Bio/Technology 8:833-839 (1990); Gordon-Kamm et al. PlantCell 2:603-618 (1990) (maize); and U.S. Pat. Nos. 5,591,616 and5,679,558 (rice).

A vector or nucleic acid construct of this invention can comprise leadersequences, transit polypeptides, promoters, terminators, genes ornucleotide sequences of interest, introns, nucleotide sequences encodinggenetic markers, etc., and any combination thereof. The nucleotidesequence(s) of the vector or nucleic acid construct can be in sense,antisense, partial antisense, or partial sense orientation in anycombination and multiple gene or nucleotide sequence copies can be used.The transgene or nucleotide sequence can come from a plant as well asfrom a non-plant source (e.g., bacteria, yeast, animals, and viruses)

A vector or nucleic acid construct comprising a transgene that is to beintroduced into a plant of this invention can comprise the transgeneand/or encoding nucleotide sequence under the control of a promoterappropriate for the expression of the transgene and/or nucleotidesequence at the desired time and/or in the desired tissue or part of theplant. Constitutive or inducible promoters can be used, as are wellknown in the art. The vector or nucleic acid construct carrying thetransgene and/or encoding nucleotide sequence can also comprise otherregulatory elements such as, e.g., translation enhancers or terminationsignals. In some embodiments, the transgene or encoding nucleotidesequence is transcribed and translated into a protein. In otherembodiments, the vector or nucleic acid construct can comprise anucleotide sequence that encodes an antisense RNA, a sense RNA that isnot translated or only partially translated, a mRNA, a tRNA, a rRNAand/or a snRNA, as are well known in the art.

E. Plant Tissue Culture and Regeneration

Plant cells, which have been transformed by any method known in the art,can also be regenerated to produce intact plants using known techniques.Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMilan Publishing Co.New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. I, 1984, and Vol. II,1986). It is known that practically all plants can be regenerated fromcultured cells or tissues.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts or a petri platecontaining transformed explants is first provided. Callus tissue isformed and shoots may be induced from callus and subsequently root.Alternatively, somatic embryo formation can be induced in the callustissue. These somatic embryos germinate as natural embryos to formplants. The culture media will generally contain various amino acids andplant hormones, such as auxin and cytokinins. A large number of plantshave been shown capable of regeneration from transformed individualcells to obtain transgenic whole plants. Patents and patent publicationscited as exemplary for the processes for transforming plant cells andregenerating plants are the following: U.S. Pat. Nos. 4,459,355,4,536,475, 5,464,763, 5,177,010, 5,187,073, 4,945,050, 5,036,006,5,100,792, 5,371,014, 5,478,744, 5,179,022, 5,565,346, 5,484,956,5,508,468, 5,538,877, 5,554,798, 5,489,520, 5,510,318, 5,204,253 and5,405,765; European Patent Nos. EP 267,159, EP 604 662, EP 672 752, EP442 174, EP 486 233, EP 486 234, EP 539 563 and EP 674 725, and PCTPublication Nos. WO 91/02071 and WO 95/06128.

The use of pollen, cotyledons, zygotic embryos, meristems and ovum asthe target tissue for transformation can eliminate or minimize the needfor extensive tissue culture work. Generally, cells derived frommeristematic tissue are useful. The method of transformation ofmeristematic cells of cereal is taught in PCT Publication No.WO96/04392. Any number of various cell lines, tissues, calli and plantparts can and have been transformed by those having knowledge in theart. Methods of preparing callus or protoplasts from various plants arewell known in the art. Cultures can be initiated from most of theabove-identified tissues. The only requirement of the plant material tobe transformed is that it can ultimately be used to produce atransformed plant.

In Duncan et al. (Planta 165:322-332 (1985)) studies were conducted thatdemonstrated that 97% of plants cultured that produced callus werecapable of plant regeneration. Subsequent experiments with both inbredsand hybrids showed that 91% appeared capable of producing regenerablecallus. In a further study (Songstad et al. Plant Cell Reports 7:262-265(1988)), several media additions were identified that enhancedregenerability of callus of two inbred lines. Other published reportsindicated that “nontraditional” tissues are capable of producing somaticembryogenesis and plant regeneration. Rao et al. (Maize GeneticsCooperation Newsletter 60:64-65 (1986)) describes somatic embryogenesisfrom glume callus cultures and Conger et al. (Plant Cell Reports6:345-347 (1987)) describes somatic embryogenesis from the tissuecultures of maize leaf segments. Thus, it is clear from the literaturethat the state of the art is such that these methods of obtaining plantsfrom callus are, and were, “conventional” in the sense that they areroutinely used and have a very high rate of success.

Tissue culture procedures of maize are described in Green and Rhodes(“Plant Regeneration in Tissue Culture of Maize” in Maize for BiologicalResearch (Plant Molecular Biology Association, Charlottesville, Va. at367 372 (1987)) and in Duncan, et al. (“The Production of Callus Capableof Plant Regeneration from Immature Embryos of Numerous Zea maysGenotypes” Planta 165: 322-332 (1985)). Thus, another aspect of thisinvention is to provide cells that upon growth and differentiationproduce maize plants having the physiological and morphologicalcharacteristics of the plants of the present invention.

Accordingly, in some embodiments, the present invention provides atissue culture of regenerable cells of HAF4561, wherein the cells of thetissue culture regenerate plants that express the genotype of HAF4561.The tissue culture can be but is not limited to tissue culture derivedfrom leaf, pollen, embryo, root, root tip, guard cell, ovule, seed,anther, silk, flower, kernel, ear, cob, husk and stalk, cell andprotoplast thereof. In some aspects of this invention, additionallyprovided is a tissue culture of regenerable cells of hybrid plantsproduced from HAF4561 germplasm. A corn plant regenerated from HAF4561or any part thereof is also included in the present invention. Thepresent invention additionally provides regenerated corn plants thatexpress the genotype of HAF4561 and/or manifest its phenotype, as wellas mutants and/or variants thereof.

F. Transgenic Plants and/or Parts Thereof of Inbred Corn Line HAF4561

The inbred corn line HAF4561 comprising at least one transgene adaptedto give HAF4561 additional and/or altered phenotypic traits is a furtheraspect of the invention. Such transgenes are often associated withregulatory elements (promoters, enhancers, terminators and the like). Asdescribed above, transgenes that can be incorporated into a plant ofthis invention include, but are not limited to, insect resistance,herbicide resistance, disease resistance, increased or decreased starchor sugars or oils, lengthened or shortened life cycle or other alteredtrait, in any combination.

In some embodiments, the present invention provides inbred corn lineHAF4561 expressing at least one transgene or nucleotide sequence adaptedto give HAF4561 modified starch traits. Further provided is the inbredcorn line HAF4561 expressing at least one mutant gene adapted to givemodified starch, fatty acid or oil traits, i.e., amylase, waxy, amyloseextender or amylose.

The present invention additionally provides the inbred corn line HAF4561and at least one transgenic gene, which can be, but is not limited to, anucleotide sequence or a synthetic sequence encoding a Bacillusthuringiensis toxin, a nucleotide sequence encoding phosphinothricinacetyl transferase (e.g., bar or pat), a nucleotide sequence encodingGdha, a nucleotide sequence encoding GOX, a nucleotide sequence encodingVIP (vegetative insect protein), a nucleotide sequence encoding aphosphomannose isomerase, nucleotide sequence encoding a FR8a (theactive insecticidal principle), a nucleotide sequence encoding EPSPsynthase, a nucleotide sequence encoding for low phytic acid production,or a nucleotide sequence encoding zein, and any combination thereof. Infurther embodiments, the present invention provides the inbred corn lineHAF4561 expressing at least one transgenic gene useful as a selectablemarker or a screenable marker, as are well known in the art.

G. Genotyping and Genetic Marker Profiles

A number of well known methods can be employed to identify the genotypeof a maize plant. One of the oldest methods is the use of isozymes,which provides a generalized footprint of the genetic material. Otherapproaches adapted to provide a higher definition profile includerestriction fragment length polymorphisms (RFLPs), amplified fragmentlength polymorphisms (AFLPs), random amplified polymorphic DNAs (RAPDs),amplification methods such as the polymerase chain reaction (PCR), whichcan employ different types of primers or probes, microsatellites (SSRs),single nucleotide polymorphisms (SNPs), sequence selection markers, etc.as are well known in the art and can be found in standard textbooks suchas Breeding Field Crops, Milton et. al. Iowa State University Press andGenetic Mapping and Marker Assisted Selection Basics, Practice andBenefits, N. Manikanda Boopathi Springer India 2013.

The marker profile of the inbred of this invention should be close tohomozygous for alleles. A marker profile produced with any of the locusidentifying systems known in the industry will identify a particularallele at a particular locus. An F1 hybrid made from the inbred of thisinvention will comprise a marker profile of the sum of both of theprofiles of its inbred parents. At each locus, the allele for the inbredof the present invention and the allele for the other inbred parentshould be present. Thus the profile of the inbred of the presentinvention allows for identification of hybrids as containing the inbredparent of the present invention. To identify the female portion of anyhybrid, the hybrid seed material from the pericarp, which is maternallyinherited, is employed in a marker technique. The resultant profile,therefore, is of the maternal parent. A comparison of this maternalprofile with the hybrid profile will allow the identification of thepaternal profile. Accordingly, some embodiments of the present inventionprovide an inbred or hybrid plant, plant part thereof, including but notlimited to a seed or an embryo, and/or a cell thereof having the allelemarker profile of the inbred plant of the this invention, HAF4561.

Marker profiles of plants of this invention can be employed to identifyessentially derived varieties or progeny developed with the inbred inits ancestry. The progeny of the inbred line of this invention, HAF4561,can be identified by identifying in the progeny the molecular markerprofile of the inbred line HAF4561, as measured by either percentidentity or percent similarity.

Different nucleotide sequences or polypeptide sequences having homologyare referred to herein as “homologues.” The term homologue includeshomologous sequences from the same and other species and orthologoussequences from the same and other species. “Homology” refers to thelevel of similarity between two or more nucleotide sequences and/oramino acid sequences in terms of percent of positional identity (i.e.,sequence similarity or identity). Therefore, as used herein “sequenceidentity” refers to the extent to which two optimally alignedpolynucleotide or polypeptide sequences are invariant throughout awindow of alignment of components, e.g., nucleotides or amino acids.“Identity” can be readily calculated by known methods including, but notlimited to, those described in: Computational Molecular Biology (Lesk,A. M., ed.) Oxford University Press, New York (1988); Biocomputing:Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NewYork (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M.,and Griffin, H. G., eds.) Humana Press, New Jersey (1994); SequenceAnalysis in Molecular Biology (von Heinje, G., ed.) Academic Press(1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J.,eds.) Stockton Press, New York (1991).

As described herein, marker systems are not just useful foridentification of the plants of this invention, but can also be used forbreeding and trait conversion techniques. Polymorphisms in maize permitthe use of markers for linkage analysis. If SSR are employed withflanking primers, the marker profile can be developed with PCR, andtherefore Southern blots can often be eliminated. Use of flankingmarkers, PCR and amplification to genotype maize is well known in theart. Primer sequences for SSR markers and maize genome mappinginformation are publicly available on the USDA website at the MaizeGenomics and Genetic Database (Maize GDB).

H. Production of Treated Seed

The present invention encompasses a method of producing treated hybridor inbred seed of the plants of the present invention and the resultanttreated seed. The method includes obtaining seed and treating the seedto improve its performance. Hybrid and inbred seed is often treated withone or more of the following including, but not limited to, fungicides,herbicides, herbicidal safeners, fertilizers, insecticides, acaricides,nematocides, bactericides, virus resistant material and/or otherbiocontrol agents. Pyrethrins, synthetic pyrethroids, oxadizinederivatives, chloronicotinyls, nitroguanidine derivatives and triazoles,organophosphates, pyrrols, pyrazoles, phenyl pyrazoles,diacylhydrazines, biological/fermentation products, carbamates and thelike are used as pesticidal seed treatments. Additionally, fludioxonil,mefenoxam, azoxystrobin, thiamethoxam, clothianidin and the like arefrequently used to treat maize seed. Methods for treating seed includebut are not limited to the use of a fluidized bed, a roller mill, arotostatic seed treater. a drum coaster, misting, soaking, filmingcoating and the like, in any combination. These methods of seedtreatment are well known in the industry.

I. Maize as Human Food and Livestock Feed

Maize is used as human food, livestock feed and as raw material inindustry. Sweet corn kernels having a relative moisture of approximately72% are consumed by humans and may be processed by canning or freezing.The food uses of maize, in addition to human consumption of maizekernels, include both products of dry- and wet-milling industries. Theprincipal products of maize dry milling are grits, meal and flour. Themaize wet-milling industry can provide maize starch, maize syrups anddextrose for food use. Maize oil is recovered from maize germ, which isa by-product of both dry- and wet-milling industries.

The present invention further encompasses a hybrid plant with a plantpart being the segregating grain formed on the ear of the hybrid. Thisgrain is a commodity plant product as are the protein concentrate,protein isolate, starch, meal, flour or oil. A number of differentindustrial processes can be employed to extract or utilize these plantproducts, as are well known in the art.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry. Industrial uses of maize include productionof ethanol, maize starch in the wet-milling industry and maize flour inthe dry-milling industry. The industrial applications of maize starchand flour are based on functional properties, such as viscosity, filmformation, adhesive properties and ability to suspend particles. Themaize starch and flour have application in the paper and textileindustries. Other industrial uses include applications in adhesives,building materials, foundry binders, laundry starches, explosives,oil-well muds, and other mining applications. Plant parts other than thegrain of maize are also used in industry: for example, stalks and husksare made into paper and wallboard and cobs are used for fuel and to makecharcoal.

The seed of the plant of the present invention can further comprise oneor more single gene traits. The plant produced from the inbred seed ofthe maize line HAF4561, the hybrid maize plant produced from thecrossing of said inbred, hybrid seed and various parts of the hybridmaize plant, can be utilized for human food, livestock feed, and as araw material in industry.

The present invention therefore also provides an agricultural productcomprising a plant of the present invention or derived from a plant ofthe present invention. The present invention further provides anindustrial product comprising a plant of the present invention orderived from a plant of the present invention. Additionally providedherein are methods of producing an agricultural and/or industrialproduct, the methods comprising planting seeds of the present invention,growing plant from such seeds, harvesting the plants and/or processingthem to obtain an agricultural or industrial product. In someembodiments, the present invention provides a method of producing acommodity plant product comprising growing the plant from the seed ofthis invention or a part thereof and producing said commodity plantproduct, wherein said commodity plant product includes, but is notlimited to, a protein concentrate, a protein isolate, starch, meal,flour, oil, or any combination thereof.

Deposit Information

Applicants have made a deposit of at least 2500 seeds of inbred cornline HAF4561 with the American Type Culture Collection (ATCC) PatentDepository, 10801 University Blvd., Manassas, Va. 20110. The ATCC numberof the deposit is PTA-120866. The date of deposit was Jan. 21, 2014, andthe seed was tested on Jan. 31, 2014 and found to be viable. Access tothis deposit will be available during the pendency of the application tothe Commissioner for Patents and persons determined by the Commissionerto be entitled thereto upon request. Upon granting of a patent on anyclaims in the application, the Applicants will make the depositavailable to the public pursuant to 37 CFR §1.808. Additionally,Applicants will meet the requirements of 37 CFR §1.801-1.809, includingproviding an indication of the viability of the sample when the depositis made. The ATCC deposit will be maintained in that depository, whichis a public depository, for a period of 30 years, or 5 years after thelast request, or for the enforceable life of the patent, whichever islonger, and will be replaced if it becomes nonviable during that period.

Additional public information on patent variety protection may beavailable from the PVP Office, a division of the U.S. Government.

VARIETY DESCRIPTION INFORMATION TABLE 1 HAF4561 VARIETY DESCRIPTIONINFORMATION #1 Type: Dent #2 Region Best Adapted: - West Central CornBelt *MG **Maturity Hybrid Group Range RM***(estimate) 4 98-102 98 #3.Brace Husk Color- Cob Color- Root Leaf Anther Glume Glume Silk After 25After 65 Line Color Color Color Color Band Color Days HSKDN Days HAF4561moderate Dark yellow greenand Pink light green tan White to Strong Greenslight purple stripes Inbred1 Moderate Dark green/ Green absent Red/light green tan Dark Green yellow Purple Reddish Orange Inbred2 moderateDark red Green and absent Yellow light green tan White to strong GreenPurple Grey Northern Southern Leaf Goss' Corn Leaf Corn Leaf LineEyespot Spot Wilt Blight Blight HAF4561 4 5 6 6 Inbred1 5 4 3 5 3Inbred2 4 4 3 4 1 PLHTN STD PLHTN ERHTN STD ERHTN Line PLHTN DEV COUNTERHTN DEV COUNT HAF4561 207.4 3.6 45 80.4 8.6 45 *MG = Maturity group**Maturity is the number of days from planting to physiological maturity(planting to black layer) ***RM = relative maturity

The data provided above is often a color. The Munsell code is areference book of color, which is known and used in the industry and bypersons with ordinary skill in the art of plant breeding. The purity andhomozygosity of inbred HAF4561 is constantly being tracked using isozymegenotypes. Isozyme data can be generated for inbred corn line HAF4561according to procedures known and published in the art.

Isozyme Genotypes for HAF4561

Isozyme data were generated for inbred corn line HAF4561 according toprocedures known and published in the art. The data in theElectrophoresis Table gives the electrophoresis data on HAF4561.

ELECTROPHORESIS RESULTS FOR HAF4561 Line PGM1 PGM2 PGD1 PGD2 IDH1 IDH2MDH1 MDH2 MDH3 MDH4 MDH5 HAF4561 9 4 3.8 2.8 4 4 6 3 16 12 12 Inbred1 94 3.8 2.8 4 4 6 3 16 12 12 Inbred2 9 4 3.8 5 4 4 6 3 16 12 12 Line MDH6ACP1 ACP4 PHI ADH HAF4561 Mm 4 2 5 4 Inbred1 Mm 2 2 5 4 Inbred2 Mm 4 2 44

The Paired Inbred Comparison Data Table A through B show a comparisonbetween HAF4561 and comparable inbreds.

PAIRED INBRED COMPARISON DATA TABLE A Inbred Yield Stand HeatUnits toP50 HeatUnits to S50 Plant Height Ear Height HAF4561 102 1397.1 1388Inbred1 91.8 1382.4 1384.3 Diff 10.2 14.6 3.6 # Expts 25 28 28 Prob0.004*** 0.023** 0.579 % Large % Large % Med. % Med. % Small % SmallInbred % Rounds % Flats Rounds Flats Rounds Flats Rounds Flats HAF4561Inbred1 34 64 Diff # Expts Prob * .05 < Prob <= .10 **.01 < Prob <= .05***.00 < Prob <= .01

In Paired Inbred Comparison Data Table B HAF4561 shows a comparison fortraits like yield, pollination, heat and silking heat units whencompared with the other inbred.

Inbred Yield Stand HeatUnits to P50 HeatUnits to S50 Plant Height EarHeight HAF4561 102 1397.1 1388 Inbred2 104.7 1461.8 1468.2 Diff 2.6 64.780.2 # Expts 25 28 28 Prob 0.45 0.000*** 0.000*** % Large % Large % Med.% Med. % Small % Small Inbred % Rounds % Flats Rounds Flats Rounds FlatsRounds Flats HAF4561 Inbred2 19.8 75.4 Diff # Expts Prob

The General Combining Ability Table shows the GCA (General CombiningAbility) estimates of HAF4561 compared with the GCA estimates of theother inbreds. The estimates show the general combining ability isweighted by the number of experiment/location combinations in which thespecific hybrid combination occurs. The interpretation of the data forall traits is that a positive comparison is a practical advantage. Anegative comparison is a practical disadvantage. The general combiningability of an inbred is clearly evidenced by the results of the generalcombining ability estimates. This data compares the inbred parent in anumber of hybrid combinations to a group of “checks”. The check data isfrom our company's and other companies' hybrids which are commercialproducts and pre-commercial hybrids, which were grown in the same setsand locations.

% Late % Early % Stalk % Push Root Root % Dropped Final Line N YieldMoisture TestWeight Lodging Test Lodging Lodging Ears Stand HAF4561 88.08 0.32 −0.72 0.31 HAF4561 99 −4.25 0.06 0.26 −1.17 −9.67 4.43 3.5 01.44 HAF4561 11 0.54 0.61 0.57 7.41 −7.35 0.27 HAF4561 13 1.93 −2.311.28 4.92 0.25 HAF4561 13 — 0.6 1.55 0.74 0 0 HAF4561 31 −1.53 −0.870.09 −0.82 1.73 −5.97 0 0.47 HAF4561 6 — 3.57 −3.42 −1.67 10.85 0.37HAF4561 29 −0.15 −0.34 −0.49 1.37 1.06 1 0 −0.29 HAF4561 28 0.14 0.19−0.44 −0.87 2.68 3.96 0.07 HAF4561 10 −0.08 0.3 1.3 7.41 −0.97 0.3HAF4561 9 −5.79 1.17 −0.31 5.1 12.78 0 HAF4561 13 −17.5 0.61 2.97 4.920.25 HAF4561 12 6.04 −0.51 −2.12 1.09 0.68 0 −1.42 HAF4561 33 8.33 −1.420.41 1.73 −0.82 9.16 0 −0.25 HAF4561 13 5.22 −2.21 −0.86 −1.47 1.93 0−0.23 HAF4561 32 16.19 −0.37 0.29 0.52 1.55 14.71 0 −0.46 HAF4561 10 —0.45 0.36 0.67 3.55 0.67 HAF4561 13 11.35 −2.11 −1.34 2.86 1.56 0 0HAF4561 31 8.03 −1.16 −0.72 1.21 0.55 14.43 0 −1.17 HAF4561 33 5.45−0.84 −0.58 1.71 1.29 15.41 0 −0.13 HAF4561 12 0.98 −0.49 −1.08 1.150.46 0 0.07 HAF4561 12 −0.94 −1.89 −1.29 0.48 1.2 0 −1.06 HAF4561 9−4.26 0.45 1.8 −1.14 0.36 HAF4561 33 7.28 0.08 0.13 2.58 0.93 8.64 0.370.53 HAF4561 8 −1.47 −1.52 −2.54 1.25 1.65 0.25 HAF4561 32 9.94 −0.72−0.41 1.52 0.97 17.57 0 −0.47 HAF4561 7 5.82 −2.8 −1.37 3.48 1.43 −11 0HAF4561 13 −0.53 0.32 0.6 0.76 1.93 0 −0.2 HAF4561 13 0.74 −2.06 −1.211.35 1.45 0 −0.28 HAF4561 8 −9.67 −1.87 0.91 2.65 −0.25 HAF4561 13 −3.830.09 0.88 3.41 0.25 HAF4561 13 10.63 −0.55 −0.23 4.92 0.25 HAF4561 262.45 0.95 −0.42 2.42 2.26 27.5 −0.06 HAF4561 13 −13.2 −0.3 0.4 4.92 0.25HAF4561 10 — −0.53 −0.21 1.65 1.68 0.13 HAF4561 20 −5.44 0.29 0.02 0.4211.95 0.07 HAF4561 13 19.68 −0.37 −2.01 −1.36 1.65 0 −0.44 HAF4561 13−11.2 −1.42 0.77 3.41 0.25 HAF4561 11 −8.07 −1.25 0.89 4.92 −1.16HAF4561 7 −12.4 −2.7 −0.93 3.48 1.43 9.71 0 HAF4561 11 6.78 0.44 0.6−1.37 1.18 0.57 HAF4561 10 −3.13 0.36 0.32 −0.29 1.03 0.16 HAF4561 8 1.4−1.81 −0.93 0 2.65 0.48 HAF4561 9 −8.3 0.8 −0.99 2.08 13.51 0.24 HAF456111 −4.34 1.91 −0.28 1.65 1.18 0.3 HAF4561 22 −1.59 −0.99 0.24 0.05 7.830 −1.39 HAF4561 12 −6.75 0.1 −0.83 0.66 −7.24 −0.98 0.13 HAF4561 12−2.37 0.81 0.48 −0.05 0.31 0 0.46 HAF4561 11 −7.7 −1.44 0.75 8.24 −1.474.8 −0.04 HAF4561 18 2.97 −0.47 1.03 4.47 50 1.44 1.47 1.81 −0.76HAF4561 13 16.45 −0.37 0.25 −1.56 3.12 0.15 HAF4561 12 −5.42 0.76 −0.061.55 0.92 0.37 0.17 HAF4561 12 — −1.03 2 4.92 0.1 HAF4561 13 −4.93 −1.730.14 3.41 0.25 HAF4561 32 10.64 −0.86 0.75 2.04 0.31 4.67 0.37 −1.05HAF4561 13 0.17 −0.8 0.37 1.49 1.72 0 0.04 HAF4561 13 −2.67 −2.3 −1.111.49 1.23 0 0.04 HAF4561 10 −9.66 −0.84 −1.32 0.29 −0.55 0.05 HAF4561 10— −0.26 0 −4.8 5.56 2.35 0 HAF4561 9 7.03 0.44 0.34 1.52 11.36 0.02HAF4561 11 −6.49 −1.21 0.08 0.13 −0.29 0.84 HAF4561 13 3.76 −0.08 −0.04−12.67 −3.53 −6.07 0.17 HAF4561 7 −1.44 −0.51 2.81 −0.18 12.83 0.36HAF4561 10 −3.11 1.75 0.39 1.75 0.07 −0.18 HAF4561 32 −2.13 −0.01 0.17−0.58 16.73 −0.98 −1.43 HAF4561 13 13.41 −0.41 −0.78 −7 −10.46 5.02−0.94 HAF4561 10 — −1.47 −4.12 1.76 −1.46 −0.64 HAF4561 10 −8.73 0.340.74 −5.88 10.15 0.16 HAF4561 11 2.66 −0.87 0 1.84 −6.25 0.93 HAF4561 12−1.16 −1.79 0.33 0.77 1.29 0.37 0.46 HAF4561 41 7.68 −0.21 −0.74 0.650.26 4.52 0.74 −0.12 HAF4561 20 −4.64 0.25 −0.15 −1.75 −4.49 −0.9HAF4561 11 1.09 0.52 1.2 2.66 1.18 0.66 HAF4561 10 −3.1 0.39 0.04 0.731.93 0.46 HAF4561 8 −1.87 2.08 −1.39 1.57 2.14 0.02 HAF4561 20 9.71 0.121.08 −4.71 1.98 −48.34 0.09 HAF4561 11 — −0.4 0.74 0.12 −6.05 5.56 0HAF4561 12 −5.81 1.99 0.6 2 1 12.29 1.16 HAF4561 7 — 5.47 0.48 0.41 0HAF4561 12 2.24 4 −0.53 2.56 1 12.29 1.16 HAF4561 9 −13 1.72 −0.5 −0.672.45 0.24 HAF4561 7 0.59 −2.13 −0.47 −8.09 17.48 0.59 HAF4561 5 1.341.33 −0.21 0 HAF4561 5 — 1.25 −2.88 0 HAF4561 11 10.22 1.51 −1.56 −0.65−0.64 4.03 0 0.36 HAF4561 20 −1.28 1.62 0.07 1.72 1.98 13.45 0.74HAF4561 7 — 4.28 −0.95 −6.76 −1.71 HAF4561 12 −8.5 3.43 0.05 0.15 −10.11.85 0.27 HAF4561 13 11.54 −0.86 −1.67 0.58 0.15 0 HAF4561 18 15.73 1.13−3.89 0.38 42.5 14.94 −0.03 HAF4561 6 9.43 −1.01 −1.43 −40 1.69 0HAF4561 13 5.45 0.36 −1.39 −0.52 0.15 0 HAF4561 11 2.03 1.98 0.44 0 4.293.61 −0.24 HAF4561 12 — 0.81 0.31 −1.47 −13.89 0.83 0.28 XR = 1424 0.13−0.14 −0.13 0.5 −5.94 2.71 3.21 0.19 0.02 XH = 94 −1.64 −0.03 −0.2 0.8120.16 1.83 3.92 0.15 0.02 XT = 14 3.4 −0.31 −0.02 0.84 −9.67 2.6 8.440.09 −0.22 XR = XH = XT = Stay- Emerg- Green % Green ence VigorHeatunits Heatunits Ear Plant Line % Snap % Barren Rating Rating to S50to P50 Height Height HAF4561 5.42 8.75 HAF4561 2.16 0.07 1.67 −0.42 0.11−26.73 −38.72 −1.69 −0.85 HAF4561 −3.02 0.5 −3.67 −3.06 HAF4561 −7.812.69 HAF4561 0 3.13 HAF4561 −1 −0.13 0.95 5.2 −36.73 −9.49 2.36 HAF4561HAF4561 −17 −0.13 1.45 −9.4 −13.93 1.36 11.62 HAF4561 −2.5 −0.8 0 1.88−2 5.19 4.46 HAF4561 −1.85 2.5 1.33 −2.39 HAF4561 HAF4561 −5.31 0.94HAF4561 10.66 11.81 HAF4561 10 −0.8 0.2 10.33 −5.73 17 14.98 HAF4561−8.5 −14.38 HAF4561 −17 −0.13 0.05 −39.47 −32.47 −1.8 2.39 HAF4561 0.376.33 −11.6 HAF4561 7.25 15.88 HAF4561 3 −0.73 −0.13 −16.47 −31.2 −3.196.4 HAF4561 −12 −0.13 −0.7 −33.47 −32.47 0.24 11.17 HAF4561 8.56 4.5HAF4561 −0.92 14.25 HAF4561 −16.81 −12.56 HAF4561 −17 0.2 −0.2 −29.13−39.13 1.88 16.46 HAF4561 −3.56 −3.25 HAF4561 −7 0.2 −0.2 14.2 1.53 5.069.21 HAF4561 0 −0.2 −5.6 18 HAF4561 −8.75 4.56 HAF4561 −2.25 8.56HAF4561 −16.13 −6.13 HAF4561 −6.31 −4.56 HAF4561 0.94 0.94 HAF4561 1.252.35 −0.25 1.06 −22.11 −22.69 3.64 −6.86 HAF4561 1.19 −0.81 HAF4561 0.88−10.6 4.4 HAF4561 2.35 −1.67 10 HAF4561 6.84 11.61 HAF4561 −10.81 −0.56HAF4561 0.19 2.44 HAF4561 1 0.8 5.4 16 HAF4561 1.25 −12.69 −10.81HAF4561 −5.98 −8.4 HAF4561 16.4 16.4 HAF4561 4.5 −11 HAF4561 0.25 −21.69−18.81 HAF4561 −0.63 −6.51 HAF4561 0.5 7.25 9.67 HAF4561 0.83 −8.83HAF4561 −1.43 −8.87 −4.4 HAF4561 4.8 0.64 0.01 2.88 −24.75 −5.17 3.83HAF4561 16.56 12.81 HAF4561 0.06 −8.19 HAF4561 −16.56 9.19 HAF4561−11.06 −4.56 HAF4561 −7 −0.13 0.3 −44.47 −21.8 1.95 4.06 HAF4561 −1.816.5 HAF4561 0.69 19.5 HAF4561 −4.81 −4.25 HAF4561 0 −1.5 −4 HAF4561 0.44−5 2.4 HAF4561 −1.75 −4.19 1.19 HAF4561 0.88 8.08 −0.58 HAF4561 HAF45610.25 −13.75 HAF4561 −4 0.91 −46.2 −39.6 0.38 −7.93 HAF4561 −0.5 12.926.75 HAF4561 −8.49 5.6 HAF4561 −17.4 −5.87 HAF4561 −7.94 −6.33 11.27HAF4561 −3.17 8.19 HAF4561 −5 −0.59 −0.47 0.45 −7.33 −23.4 −9.93 1.54HAF4561 −0.81 −5.47 −8.79 HAF4561 0.25 −3.69 −0.31 HAF4561 −2.5 −6.383.09 HAF4561 1.91 −5 −3.1 HAF4561 −10 −0.8 0.7 19.67 3.6 −4.87 13.53HAF4561 9.69 −6.25 HAF4561 0.33 16.83 HAF4561 −0.13 −9.5 HAF4561 12.8319.33 HAF4561 5.25 13 HAF4561 4.75 −7.5 HAF4561 −4.38 HAF4561 −14.38HAF4561 6.67 12.5 HAF4561 0 −0.13 0.2 −7.33 −5.73 13.8 12.2 HAF4561−5.13 −14.5 HAF4561 −3.72 −5.42 HAF4561 11.25 21 HAF4561 0 1 −30.75−44.83 7.83 −1.92 HAF4561 HAF4561 −1.25 −4 HAF4561 1 −2.5 HAF4561 13.57.5 XR = −4.38 0.03 0.25 −0.26 0.26 −15.51 −24.24 −1.11 2.71 XH = −5.19−0.24 −0.17 −0.17 0.35 −14.37 −22.78 −0.88 2.35 XT = −5.22 0.07 0.54−0.29 0.3 −17.37 −24.17 −1.11 3.08 XR = XH = XT = Line = inbred which iscrossed to second parents to produce results XR = GCA Estimate: Weightedby Expt XH = GCA Estimate: Weighted by Parent2 XT = Same as XH but usingonly those parent2 with two years of dataThe Paired Hybrid Comparison Data Table A through B shows the inbredHAF4561 in hybrid combination, as Hybrid 1, in comparison with anotherhybrid, which is adapted for the same region of the Corn Belt.

PAIRED HYBRID COMPARISON DATA TABLE A Hybrid Yield Moist TWT PCTERLPCTSL PCTPUSH PLTLRL PCTDE Hybrid1 w/HAF4561 164.7 19.2 56.7 0.4 2.429.6 1.6 0 Hybrid2 168.9 19.3 56.4 1.5 1.4 34.6 7.7 0 # Expts 68 68 54 327 14 14 3 Diff 4.2 0.2 0.3 1.1 1 5 6.1 0 Prob 0.108 0.579 0.213 0.1850.342 0.364 0.129 Hybrid Stand PCTSG PCTGS PctBarren Emerge Vigor HUS50Hybrid1 w/HAF4561 226.7 25 0.6 3.9 3.4 3.2 1211 Hybrid2 227.2 18.3 0 5.12.9 3 1196 # Expts 68 3 2 5 25 26 6 Diff 0.5 6.7 0.6 1.2 0.5 0.1 15.2Prob 0.648 0.27 0.5 0.416 0.083* 0.64 0.111 Hybrid HUP50 Pltht EarhtHybrid1 w/HAF4561 1199 247.1 115.8 Hybrid2 1195 249.4 106 # Expts 6 6 6Diff 4.8 2.3 9.8 Prob 0.546 0.748 0.344 Hybrid Yield Moist TWT PCTERLPCTSL PCTPUSH PLTLRL PCTDE Hybrid1 w/HAF4561 169.5 19.1 56.9 3.2 3.130.3 1.7 0 Hybrid3 169.9 19.2 57.4 3.7 3.6 17 13.1 0 # Expts 86 86 66 638 15 20 4 Diff 0.4 0.2 0.5 0.5 0.4 13.3 11.4 0 Prob 0.871 0.49 0.016**0.882 0.726 0.061* 0.016** Hybrid Stand PCTSG PCTGS PctBarren EmergeVigor HUS50 Hybrid1 w/HAF4561 193.9 25 0.4 3.9 3.6 3.5 1225 Hybrid3 18416.7 0 7.5 3.3 2.8 1291 # Expts 86 3 3 5 29 34 8 Diff 10 8.3 0.4 3.6 0.20.7 66.1 Prob 0.000*** 0.588 0.423 0.346 0.243 0.005*** 0.000*** HybridHUP50 Pltht Earht Hybrid1 w/HAF4561 1213 248.5 115.8 Hybrid3 1298 263.3132.2 # Expts 8 8 8 Diff 84.6 14.8 16.3 Prob 0.000*** 0.074* 0.025***.05 < Prob <= .10 **.01 < Prob <= .05 ***.00 < Prob <= .01The Yield by Environment Response Table shows the yield response ofHybrid 1 w/HAF4561 as a parent in comparison with two other hybrids andthe plants in the environment around it at the same location.

YIELD BY ENVIRONMENT RESPONSE TABLE Research Plots # Environment YieldHybrid Error Plots 75 100 125 150 175 200 Hybrid1 20.6 107 67 93 120 147173 200 w/HAF4561 Hybrid2 22.5 167 75 100 125 150 175 200 Hybrid1 20.6107 67 93 120 147 173 200 w/HAF4561 Hybrid3 22.8 746 70 96 123 149 176203 Hybrid1 20.6 107 67 93 120 147 173 200 w/HAF4561 Hybrid5 23.5 196578 104 130 155 181 206

Accordingly, the present invention has been described with some degreeof particularity directed to the embodiment of the present invention. Itshould be appreciated, though that the present invention is defined bythe following claims construed in light of the prior art so thatmodifications or changes may be made to the embodiment of the presentinvention without departing from the inventive concepts containedherein.

The invention claimed is:
 1. A seed of maize variety HAF4561, whereinrepresentative seed of said variety HAF4561 have been deposited underATCC Accession Number PTA-120866.
 2. A plant of maize variety HAF4561,wherein representative seed of said variety HAF4561 have been depositedunder ATCC Accession Number PTA-120866.
 3. A plant part of the plant ofclaim
 2. 4. The plant part of claim 2, wherein said part is a pollengrain, a silk, a protoplast, a cell, a tassel, an anther or an ovule. 5.A maize seed produced on the plant of claim
 2. 6. A maize plant havingall the physiological and morphological characteristics of the plantaccording to claim 2 and further comprising an additional trait, whereinthe additional trait is selected from the group consisting of increasedwater stress resistance, waxy starch, male sterility or restoration ofmale fertility, modified carbohydrate metabolism, modified proteinmetabolism and modified fatty acid metabolism, altered starch,thermotolerant amylase, herbicide resistance, insect resistance,nematode resistance, bacterial disease resistance, fungal diseaseresistance, and viral disease resistance.
 7. The plant of claim 6wherein the additional trait is conferred by introducing a transgene. 8.A process for producing maize seed, said process comprising crossing themaize plant of claim 2 with a different maize plant to produce an F1hybrid seed.
 9. A maize seed produced by the process of claim
 8. 10. Amaize plant produced by germinating the seed of claim
 9. 11. A method ofproducing a genetic marker profile comprising extracting nucleic acidsfrom the seed of claim 9 or the plant germinated from said seed andgenotyping said nucleic acids at one or more genetic loci, therebyproducing a genetic marker profile.
 12. A method of plant breedingcomprising a)isolating nucleic acids from the seed of claim 9, b)identifying one or more polymorphisms from the isolated nucleic acids,and c) selecting a plant having one or more polymorphisms wherein theplant is used in a plant breeding method.
 13. A method of plant breedingcomprising a) isolating nucleic acids from the plant of claim 10, b)identifying one or more polymorphisms from the isolated nucleic acids,and c) selecting a plant having one or more polymorphisms wherein theplant is used in a plant breeding method.
 14. A process of introducingan additional trait into maize plant HAF4561 comprising: (a) crossingHAF4561 plants grown from HAF4561 seed, representative seed depositedunder ATCC Accession Number PTA-120866, with plants of another maizeplant that comprise an additional trait to produce hybrid progenyplants, (b) selecting hybrid progeny plants that have the additionaltrait to produce selected hybrid progeny plants; (c) crossing theselected progeny plants with the HAF4561 plants to produce backcrossprogeny plants; (d) selecting for backcross progeny plants that have theadditional trait to produce selected backcross progeny plants; and (e)repeating steps (c) and (d) at least three or more times to producebackcross progeny plants that comprise the additional trait and all ofthe physiological and morphological characteristics of maize inbredplant HAF4561 when grown in the same environmental conditions.
 15. Aplant produced by the process of claim
 14. 16. A method of producing amaize plant derived from the inbred plant HAF4561, the method comprisingthe steps of (a) growing the plant of claim 10; (b) crossing said plantwith itself or a different plant to produce a seed of a progeny plant;(c) growing said progeny plant from said seed and crossing the progenyplant with itself or a different plant to produce an inbred maize plantderived from the inbred plant HAF4561.
 17. A method for developing asecond maize plant in a maize plant breeding program, comprisingapplying plant breeding techniques wherein said techniques compriserecurrent selection, backcrossing, pedigree breeding, marker enhancedselection, haploid/double haploid production, or transformation to themaize plant of claim 10, or its parts, wherein application of saidtechniques results in development of a second maize variety.
 18. Amethod of producing a commodity plant product comprising growing theplant from the seed of claim 9, or a part thereof, and producing saidcommodity plant product comprising protein concentrate, protein isolate,starch, meal, flour or oil therefrom.
 19. A method of producing a maizeplant with doubled haploid chromosomes derived from the maize varietyHAF4561 the method comprising: (a) crossing the plant of claim 10,wherein said plant may have one or more traits, with an inducer maizeplant to produce a progeny with haploid chromosomes; and (b) doublingthe haploid chromosomes in the progeny to produce a maize plant withdoubled haploid chromosomes.